The H2O2-sensitive HyPer protein targeted to the endoplasmic reticulum as a mirror of the oxidizing thiol-disulfide milieu

Oxidative protein folding in the endoplasmic reticulum (ER) is associated with the formation of native disulfide bonds, which inevitably results in the formation of hydrogen peroxide (H2O2). Particularly in pancreatic p-cells with their high secretory activity and extremely low antioxidant capacity, the H2O2 molecules generated during oxidative protein folding could represent a significant oxidative burden. Therefore this study was conducted to elucidate the H2O2 generation during disulfide bond formation in insulin-producing RINm5F cells by targeting and specifically expressing the H2O2-sensitive biosensor HyPer in the ER (ER-HyPer). In addition the influence of overexpression of the H2O2-metabolizing ER-resident peroxiredoxin IV (PRDXIV) on H2O2 levels was examined. The ER-HyPer fluorescent protein was completely oxidized, whereas HyPer expressed in cytosol, peroxisomes, and mitochondria was prevalently in the reduced state, indicating a high basal H2O2 concentration in the ER lumen. These results could also be confirmed in non-insulin-producing COS-7 cells. Overexpression of PRDXIV in RINm5F cells effectively protected against H2O2-mediated toxicity: however, it did not affect the fluorescence signal of ER-HyPer. Moreover, the inhibition of de novo protein synthesis and the associated H2O2 generation by cycloheximide had no influence on the ER-HyPer redox state. Taken together, these findings strongly suggest that the H2O2-sensitive biosensor reflects exclusively the oxidative milieu in the ER and not the H2O2 concentration in the ER lumen. (C) 2012 Elsevier Inc. All rights reserved.