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dc.contributor.authorMelo, EPen
dc.contributor.authorLopes, Cen
dc.contributor.authorGollwitzer, Pen
dc.contributor.authorLortz, Sen
dc.contributor.authorLenzen, Sen
dc.contributor.authorMehmeti, Ien
dc.contributor.authorKaminski, Clemensen
dc.contributor.authorRon, Daviden
dc.contributor.authorAvezov, Edwarden
dc.date.accessioned2017-05-30T16:26:48Z
dc.date.available2017-05-30T16:26:48Z
dc.date.issued2017-03-27en
dc.identifier.issn1741-7007
dc.identifier.urihttps://www.repository.cam.ac.uk/handle/1810/264500
dc.description.abstract$\textbf{Background:}$ The fate of hydrogen peroxide (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. Over the years hints have suggested that glutathione, puzzlingly abundant in the ER lumen, might have a role in reducing the heavy burden of H$_2$O$_2$ produced by the luminal enzymatic machinery for disulfide bond formation. However, limitations in existing organelle-targeted H$_2$O$_2$ probes have rendered them inert in the thiol-oxidizing ER, precluding experimental follow-up of glutathione’s role in ER H$_2$O$_2$ metabolism. $\textbf{Results:}$ 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 the lumen of the ER further enhanced the luminal H$_2$O$_2$ signal and eroded β-cell viability. $\textbf{Conclusions:}$ A tri-cysteine system with a single peroxidatic thiol enables H$_2$O$_2$ detection in oxidizing milieux such as that of the ER. Tracking ER H$_2$O$_2$ in live pancreatic β-cells points to a role for glutathione in H$_2$O$_2$ turnover.
dc.description.sponsorshipThis work is supported by grants from the Wellcome Trust (Wellcome 200848/Z/16/Z, WT: UNS18966), Fundação para a Ciência e Tecnologia, Portugal (PTDC/QUI/BIQ/119677/2010 and UID/BIM/04773/2013-CBMR), European Commission (EU FP7 Beta-Bat No: 277713), EPSRC (1503478), MRC (MR/K015850/1), and a Wellcome Trust Strategic Award for core facilities to the Cambridge Institute for Medical Research (Wellcome 100140). DR is a Wellcome Trust Principal Research Fellow.
dc.languageengen
dc.language.isoenen
dc.publisherBioMed Central
dc.rightsAttribution 4.0 Internationalen
dc.rightsAttribution 4.0 Internationalen
dc.rightsAttribution 4.0 Internationalen
dc.rightsAttribution 4.0 Internationalen
dc.rightsAttribution 4.0 Internationalen
dc.rights.urihttp://creativecommons.org/licenses/by/4.0/en
dc.rights.urihttp://creativecommons.org/licenses/by/4.0/en
dc.rights.urihttp://creativecommons.org/licenses/by/4.0/en
dc.rights.urihttp://creativecommons.org/licenses/by/4.0/en
dc.rights.urihttp://creativecommons.org/licenses/by/4.0/en
dc.subjectendoplasmic reticulumen
dc.subjectfluorescence lifetime imagingen
dc.subjectfluorescent protein sensoren
dc.subjectglutathioneen
dc.subjectH2O2 probeen
dc.subjecthydrogen peroxideen
dc.subjectlive cell imagingen
dc.subjectpancreatic β-cellsen
dc.subjectredoxen
dc.titleTriPer, an optical probe tuned to the endoplasmic reticulum tracks changes in luminal H$_2$O$_2$en
dc.typeArticle
prism.issueIdentifier1en
prism.number24en
prism.publicationDate2017en
prism.publicationNameBMC Biologyen
prism.volume15en
dc.identifier.doi10.17863/CAM.10049
dcterms.dateAccepted2017-03-14en
rioxxterms.versionofrecord10.1186/s12915-017-0367-5en
rioxxterms.versionVoRen
rioxxterms.licenseref.urihttp://creativecommons.org/licenses/by/4.0/en
rioxxterms.licenseref.startdate2017-03-27en
dc.contributor.orcidKaminski, Clemens [0000-0002-5194-0962]
dc.contributor.orcidRon, David [0000-0002-3014-5636]
dc.contributor.orcidAvezov, Edward [0000-0002-2894-0585]
dc.identifier.eissn1741-7007
rioxxterms.typeJournal Article/Reviewen
pubs.funder-project-idMRC (MR/K015850/1)
pubs.funder-project-idWELLCOME TRUST (200848/Z/16/Z)
pubs.funder-project-idBBSRC (BB/H023917/1)
pubs.funder-project-idEPSRC (EP/H018301/1)
pubs.funder-project-idMRC (MR/K02292X/1)
pubs.funder-project-idMRC (G0902243)
pubs.funder-project-idWellcome Trust (100140/Z/12/Z)
pubs.funder-project-idEC FP7 CP (277713)
cam.orpheus.successThu Jan 30 12:53:52 GMT 2020 - The item has an open VoR version.*
rioxxterms.freetoread.startdate2100-01-01


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Attribution 4.0 International
Except where otherwise noted, this item's licence is described as Attribution 4.0 International