TriPer, an optical probe tuned to the endoplasmic reticulum tracks changes in luminal H₂O₂

The fate of hydrogen peroxide (H₂O₂) 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. Over the years hints have suggested that glutathione, puzzlingly abundant in the ER lumen, might have a role in reducing the heavy burden of H₂O₂ produced by the luminal enzymatic machinery for disulfide bond formation. However, limitations in existing organelle-targeted H₂O₂ probes have rendered them inert in the thiol-oxidizing ER, precluding experimental follow-up of glutathione's role in ER H₂O₂ metabolism.

Here we report on the development of TriPer, a vital optical probe sensitive to changes in the concentration of H₂O₂ in the thiol-oxidizing environment of the ER. Consistent with the hypothesized contribution of oxidative protein folding to H₂O₂ production, ER-localized TriPer detected an increase in the luminal H₂O₂ 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 the lumen of the ER further enhanced the luminal H₂O₂ signal and eroded β-cell viability.

A tri-cysteine system with a single peroxidatic thiol enables H₂O₂ detection in oxidizing milieux such as that of the ER. Tracking ER H₂O₂ in live pancreatic β-cells points to a role for glutathione in H₂O₂ turnover.