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BMC Biol. 2017 Mar 27;15(1):24. doi: 10.1186/s12915-017-0367-5.

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

Author information

1
University of Cambridge, Cambridge Institute for Medical Research, the Wellcome Trust MRC Institute of Metabolic Science and NIHR Cambridge Biomedical Research Centre, Cambridge Biomedical Campus, Hills Road, Cambridge, CB2 0XY, UK.
2
Centre for Biomedical Research, Universidade do Algarve, Faro, Portugal.
3
Institute of Clinical Biochemistry, Hannover Medical School, Hannover, 30625, Germany.
4
Department of Chemical Engineering and Biotechnology, University of Cambridge, Cambridge, CB2 3RA, UK.
5
University of Cambridge, Cambridge Institute for Medical Research, the Wellcome Trust MRC Institute of Metabolic Science and NIHR Cambridge Biomedical Research Centre, Cambridge Biomedical Campus, Hills Road, Cambridge, CB2 0XY, UK. dr360@medschl.cam.ac.uk.
6
University of Cambridge, Cambridge Institute for Medical Research, the Wellcome Trust MRC Institute of Metabolic Science and NIHR Cambridge Biomedical Research Centre, Cambridge Biomedical Campus, Hills Road, Cambridge, CB2 0XY, UK. ea347@cam.ac.uk.

Abstract

BACKGROUND:

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.

RESULTS:

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.

CONCLUSIONS:

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.

KEYWORDS:

Endoplasmic reticulum; Fluorescence lifetime imaging; Fluorescent protein sensor; Glutathione; H2O2 probe; Hydrogen peroxide; Live cell imaging; Pancreatic β-cells; Redox

PMID:
28347335
PMCID:
PMC5368998
DOI:
10.1186/s12915-017-0367-5
[Indexed for MEDLINE]
Free PMC Article

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