Balanced Regulation of Redox Status of Intracellular Thioredoxin Revealed by in-Cell NMR

To understand how intracellular proteins respond to oxidative stresses, the redox status of the target protein, as well as the intracellular redox potential (<i>E</i>_GSH), which is defined by the concentrations of reduced and oxidized glutathione, should be observed simultaneously within living cells. In this study, we developed a method that can monitor the redox status of thioredoxin (Trx) and <i>E</i>_GSH by direct NMR observation of Trx and glutathione within living cells. Unlike the midpoint potential of Trx measured <i>in vitro</i> (∼ –300 mV), the intracellular Trx exhibited the redox transition at <i>E</i>_GSH between −250 and −200 mV, the range known to trigger the oxidative stress-mediated signalings. Furthermore, we quantified the contribution of Trx reductase to the redox status of Trx, demonstrating that the redox profile of Trx is determined by the interplay between the elevation of <i>E</i>_GSH and the reduction by Trx reductase and other endogenous molecules