title: TriPer, an optical probe tuned to the endoplasmic reticulum tracks changes in luminal H2O2.
abstract: 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 H2O2produced by the luminal enzymatic machinery for disulfide bond formation. However, limitations in existing organelle-targeted H2O2probes have rendered them inert in the thiol-oxidizing ER, precluding experimental follow-up of glutathione's role in ER H2O2metabolism.
  
  
  
  Here we report on the development of TriPer, a vital optical probe sensitive to changes in the concentration of H2O2in the thiol-oxidizing environment of the ER. Consistent with the hypothesized contribution of oxidative protein folding to H2O2production, ER-localized TriPer detected an increase in the luminal H2O2signal 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 H2O2signal and eroded β-cell viability.
  
  
  
  A tri-cysteine system with a single peroxidatic thiol enables H2O2detection in oxidizing milieux such as that of the ER. Tracking ER H2O2in live pancreatic β-cells points to a role for glutathione in H2O2turnover.
authors.affiliation: 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.|Centre for Biomedical Research, Universidade do Algarve, Faro, Portugal.
authors.forename: Eduardo Pinho
authors.initials: EP
authors.lastname: Melo
authors.affiliation: Centre for Biomedical Research, Universidade do Algarve, Faro, Portugal.
authors.forename: Carlos
authors.initials: C
authors.lastname: Lopes
authors.affiliation: 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.
authors.forename: Peter
authors.initials: P
authors.lastname: Gollwitzer
authors.affiliation: Institute of Clinical Biochemistry, Hannover Medical School, Hannover, 30625, Germany.
authors.forename: Stephan
authors.initials: S
authors.lastname: Lortz
authors.affiliation: Institute of Clinical Biochemistry, Hannover Medical School, Hannover, 30625, Germany.
authors.forename: Sigurd
authors.initials: S
authors.lastname: Lenzen
authors.affiliation: Institute of Clinical Biochemistry, Hannover Medical School, Hannover, 30625, Germany.
authors.forename: Ilir
authors.initials: I
authors.lastname: Mehmeti
authors.affiliation: Department of Chemical Engineering and Biotechnology, University of Cambridge, Cambridge, CB2 3RA, UK.
authors.forename: Clemens F
authors.initials: CF
authors.lastname: Kaminski
authors.affiliation: 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.
authors.forename: David
authors.initials: D
authors.lastname: Ron
authors.affiliation: 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.
authors.forename: Edward
authors.identifier: 0000-0002-2894-0585
authors.initials: E
authors.lastname: Avezov
mesh_terms: D000818:Animals; D002384:Catalysis; D002460:Cell Line; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism*; D005453:Fluorescence; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism* / Q000494:pharmacology; D050417:Insulin-Secreting Cells / Q000187:drug effects / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D015335:Molecular Probes / Q000378:metabolism*; D055620:Optical Phenomena; D010084:Oxidation-Reduction; D013438:Sulfhydryl Compounds / Q000378:metabolism
chemical_list: D015335:Molecular Probes; D013438:Sulfhydryl Compounds; D006861:Hydrogen Peroxide; D005978:Glutathione
keywords: Endoplasmic reticulum; Fluorescence lifetime imaging; Fluorescent protein sensor; Glutathione; H2O2 probe; Hydrogen peroxide; Live cell imaging; Pancreatic β-cells; Redox