A novel tag-free probe for targeting molecules interacting with a flavonoid catabolite

Abstract

3,4-Dihydroxyphenylacetic acid (DOPAC) is one of the colonic microflora-produced catabolites of quercetin 4′-glucoside (Q4′G). Although the interaction of DOPAC with cellular proteins might be involved in its biological activity, the actual proteins have not yet been identified. In this study, we developed a novel tag-free DOPAC probe to label the targeted proteins by the copper(I)-catalyzed azide alkyne cycloaddition (CuAAC) and verified its efficacy. Various labeled proteins were detected by the DOPAC probe with the azide labeled biotin and a horseradish peroxidase (HRP)-streptavidin complex. Furthermore, a pulldown assay identified Keap1 and aryl hydrocarbon receptor (AhR) as the target proteins for the phase 2 enzyme up-regulation.

3,4-Dihydroxyphenylacetic acid is a potential aldehyde dehydrogenase inducer in murine hepatoma Hepa1c1c7 cells

“…24) Our group has successfully detected the direct modification of Keap1 and AhR by DOPAC using its click chemistry probe and the pull down assay. 15) These results indicated that covalent modification of Keap1 and AhR by DOPAC might be involved in the activation of these pathways and upregulation of ALDH genes in Hepa1c1c7 cells. DOPAC not only induced the gene expression of ALDHs, but also the phase 2 genes including glutathione S-transferase, 25) heme oxygenase-1 and glutamate-cysteine ligase, catalytic subunit, 6) both of which play an effective role in protection against oxidative stress.…”

“…21) AhR has been identified as a potential target of covalent modification by DOPAC. 15) Expectedly, DOPAC significantly increased the nuclear level of the AhR proteins (Fig. 3(A) and (B)).…”

“…A previous study using a DOPAC click chemistry probe indicated that the signaling pathway of Nrf2 and/ or AhR is attributable to the gene expression of the phase 2 drug-metabolizing enzymes. 6,15) NF-κB is known as the nuclear factor that interacts positively or negatively with Nrf2 and AhR. 16) To investigate whether Nrf2, AhR and NF-κB, are involved in the ALDH expression, we examined their protein expression.…”

Flavonoid metabolism: the interaction of metabolites and gut microbiota

“…Similar to the bacterial metabolism of daidzein, sesame lignans and ellagitannins are converted into enterolactone / enterodiol and urolithin A, respectively, all of which can bind to estrogen receptors [59]. Ring-fission catabolism has been observed in the case of tea catechins, the primary class of flavonoids in green tea; γ-valerolactones have been identified in human urine after green tea ingestion ( Figure 2 targets of covalent modification by DOPAC in mouse hepatoma cells [77]. Thus, even though DOPAC shows relatively weaker biological activities than quercetin aglycon [72,76], DOPAC is quite plausibly a main component that facilitates the health-promoting potential of quercetin glycosides.…”

Covalent adduction of endogenous and food-derived quinones to a protein: its biological significance

“…3,4-Dihydroxyphenylacetic acid, a breakdown product from quercetin glycosides, could be absorbed into the circulation and show biological activities through electrophilic adduction of the corresponding quinone to biomolecules. ( 19 )…”