TriPer, an optical probe tuned to the endoplasmic reticulum tracks H2O2 consumption by glutathione

Abstract:

The fate of H 2 O 2 in the endoplasmic reticulum (ER) has been inferred indirectly from the activity of ER localized thiol oxidases and peroxiredoxins, in vitro , and the consequences of their genetic manipulation, in vivo . Here we report on the development of TriPer, a vital optical probe sensitive to changes in the concentration of H 2 O 2 in the thiol-oxidizing environment of the ER. Consistent with the hypothesized contribution of oxidative protein folding to H 2 O 2 production, ER-localized TriPer detected an increase in the luminal H 2 O 2 signal upon induction of pro-insulin (a disulfide bonded protein of pancreatic β-cells), which was attenuated by the ectopic expression of catalase in the ER lumen. Interfering with glutathione production in the cytosol by buthionine sulfoximine (BSO) or enhancing its localized destruction by expression of the glutathione-degrading enzyme ChaC1 in lumen of the ER, enhanced further the luminal H 2 O 2 signal and eroded β-cell viability. Tracking ER H 2 O 2 in live cells points to an unanticipated role for glutathione in H 2 O 2 turnover. Significance statement The presence of millimolar glutathione in the lumen of the endoplasmic reticulum has been difficult to understand purely in terms of modulation of protein-based disulphide bond formation in secreted proteins. Over the years hints have suggested that glutathione might have a role in reducing the heavy burden of hydrogen peroxide (H 2 O 2 ) produced by the luminal enzymatic machinery for disulphide bond formation. However, limitations in existing in vivo H 2 O 2 probes have rendered them all but useless in the thiol-oxidizing ER, precluding experimental follow-up of glutathione’s role ER H 2 O 2 metabolism. Here we report on the development and mechanistic characterization of an optical probe, TriPer that circumvents the limitations of previous sensors by retaining specific responsiveness to H 2 O 2 in thiol-oxidizing environments. Application of this tool to the ER of an insulin-producing pancreatic b-cells model system revealed that ER glutathione antagonizes locally-produced H 2 O 2 resulting from the oxidative folding of pro-insulin. This study presents an interdisciplinary effort intersecting cell biology and chemistry: An original redox chemistry concept leading to development of a biological tool, broadly applicable for in vivo studies of H 2 O 2 metabolism in the ER. More broadly, the concept developed here sets a precedent for applying a tri-cysteine relay system to discrimination between various oxidative reactants, in complex redox milieux.