A screening system for antioxidants using thioredoxin-deficient yeast: discovery of thermostable antioxidant activity from Agaricus blazei Murill.

Abstract

Previously, we found that cytosolic thioredoxin is a negative regulator for an oxidative stress responsive transcription factor, Yap1p (yeast AP-1-like transcription factor), i.e., this transcription factor is constitutively concentrated in the nucleus in the thioredoxin-deficient mutant ( trx1delta trx2delta) due to an impairment of the reactive oxygen species-scavenging activity of this mutant [Izawa et al. (1999) J Biol Chem 274:28459-28465]. Based on these findings, we developed a screening method to discover substances that show antioxidant activity. With this method, antioxidant activity was evaluated by monitoring the subcellular localization of Yap1p. Since Yap1p is oxidized and accumulates in the nucleus in trx1delta trx2Delta cells, it is easy to identify antioxidant activity by observing the localization of green fluorescent protein (GFP)-tagged Yap1p. If exogenous substances taken in by trx1delta trx2Delta cells were able to function as antioxidants to reduce the oxidized form of Yap1p, GFP1-Yap1p would diffuse into the cytoplasm. We used this system to screen for antioxidant activity in mushrooms, and found that the edible mushroom Agaricus blazei Murill is an excellent source of antioxidants.