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

BMC Biology

Research Article

Carlos Lopes¹  Clemens F. Kaminski²  Sigurd Lenzen³  Stephan Lortz³  Ilir Mehmeti³  Edward Avezov⁴  David Ron⁴  Peter Gollwitzer⁴  Eduardo Pinho Melo⁵ 

[1] Centre for Biomedical Research, Universidade do Algarve, Faro, Portugal;Department of Chemical Engineering and Biotechnology, University of Cambridge, CB2 3RA, Cambridge, UK;Institute of Clinical Biochemistry, Hannover Medical School, 30625, Hannover, Germany;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, CB2 0XY, Cambridge, UK;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, CB2 0XY, Cambridge, UK;Centre for Biomedical Research, Universidade do Algarve, Faro, Portugal;

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

DOI  :  10.1186/s12915-017-0367-5

 received in 2017-02-28, accepted in 2017-03-14,  发布年份 2017

来源: Springer

【 摘 要 】

BackgroundThe 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.ResultsHere 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.ConclusionsA 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.

【 授权许可】

CC BY   
© Avezov et al. 2017