TriPer, an optical probe tuned to the endoplasmic reticulum tracks changes in luminal H2O2
Autor(a) principal: | |
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Data de Publicação: | 2012 |
Outros Autores: | , , , , , , , |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
Texto Completo: | http://hdl.handle.net/10400.1/11538 |
Resumo: | 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 beta-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 beta-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 beta-cells points to a role for glutathione in H2O2 turnover. |
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TriPer, an optical probe tuned to the endoplasmic reticulum tracks changes in luminal H2O2Disulfide-Bond FormationEnzyme Gene-ExpressionHydrogen-PeroxideThiol-RedoxBeta-CellGlutathioneProteinPeroxiredoxinStressPoiseBackground: 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 beta-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 beta-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 beta-cells points to a role for glutathione in H2O2 turnover.Wellcome Trust [Wellcome 200848/Z/16/Z, WT: UNS18966]; Fundacao para a Ciencia e Tecnologia, Portugal [PTDC/QUI/BIQ/119677/2010, UID/BIM/04773/2013-CBMR]; European Commission (FU FP7 Beta-Bat) [277713]; EPSRC [1503478]; MRC [MR/K015850/1]; Wellcome Trust Strategic Award for core facilities to the Cambridge Institute for Medical Research [Wellcome 100140]; Wellcome Trust Principal Research FellowBiomed Central LtdSapientiaMelo, EduardoRafael dos Reis Lopes, CarlosGollwitzer, PeterLortz, StephanLenzen, SigurdMehmeti, IlirKaminski, Clemens F.Ron, DavidAvezov, Edward2018-12-07T14:53:29Z2012-062012-06-01T00:00:00Zinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articleapplication/pdfhttp://hdl.handle.net/10400.1/11538eng1741-700710.1186/s12915-017-0367-5info:eu-repo/semantics/openAccessreponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãoinstacron:RCAAP2023-07-24T10:23:22Zoai:sapientia.ualg.pt:10400.1/11538Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireopendoar:71602024-03-19T20:03:01.936569Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãofalse |
dc.title.none.fl_str_mv |
TriPer, an optical probe tuned to the endoplasmic reticulum tracks changes in luminal H2O2 |
title |
TriPer, an optical probe tuned to the endoplasmic reticulum tracks changes in luminal H2O2 |
spellingShingle |
TriPer, an optical probe tuned to the endoplasmic reticulum tracks changes in luminal H2O2 Melo, Eduardo Disulfide-Bond Formation Enzyme Gene-Expression Hydrogen-Peroxide Thiol-Redox Beta-Cell Glutathione Protein Peroxiredoxin Stress Poise |
title_short |
TriPer, an optical probe tuned to the endoplasmic reticulum tracks changes in luminal H2O2 |
title_full |
TriPer, an optical probe tuned to the endoplasmic reticulum tracks changes in luminal H2O2 |
title_fullStr |
TriPer, an optical probe tuned to the endoplasmic reticulum tracks changes in luminal H2O2 |
title_full_unstemmed |
TriPer, an optical probe tuned to the endoplasmic reticulum tracks changes in luminal H2O2 |
title_sort |
TriPer, an optical probe tuned to the endoplasmic reticulum tracks changes in luminal H2O2 |
author |
Melo, Eduardo |
author_facet |
Melo, Eduardo Rafael dos Reis Lopes, Carlos Gollwitzer, Peter Lortz, Stephan Lenzen, Sigurd Mehmeti, Ilir Kaminski, Clemens F. Ron, David Avezov, Edward |
author_role |
author |
author2 |
Rafael dos Reis Lopes, Carlos Gollwitzer, Peter Lortz, Stephan Lenzen, Sigurd Mehmeti, Ilir Kaminski, Clemens F. Ron, David Avezov, Edward |
author2_role |
author author author author author author author author |
dc.contributor.none.fl_str_mv |
Sapientia |
dc.contributor.author.fl_str_mv |
Melo, Eduardo Rafael dos Reis Lopes, Carlos Gollwitzer, Peter Lortz, Stephan Lenzen, Sigurd Mehmeti, Ilir Kaminski, Clemens F. Ron, David Avezov, Edward |
dc.subject.por.fl_str_mv |
Disulfide-Bond Formation Enzyme Gene-Expression Hydrogen-Peroxide Thiol-Redox Beta-Cell Glutathione Protein Peroxiredoxin Stress Poise |
topic |
Disulfide-Bond Formation Enzyme Gene-Expression Hydrogen-Peroxide Thiol-Redox Beta-Cell Glutathione Protein Peroxiredoxin Stress Poise |
description |
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 beta-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 beta-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 beta-cells points to a role for glutathione in H2O2 turnover. |
publishDate |
2012 |
dc.date.none.fl_str_mv |
2012-06 2012-06-01T00:00:00Z 2018-12-07T14:53:29Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/article |
format |
article |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
http://hdl.handle.net/10400.1/11538 |
url |
http://hdl.handle.net/10400.1/11538 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
1741-7007 10.1186/s12915-017-0367-5 |
dc.rights.driver.fl_str_mv |
info:eu-repo/semantics/openAccess |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
application/pdf |
dc.publisher.none.fl_str_mv |
Biomed Central Ltd |
publisher.none.fl_str_mv |
Biomed Central Ltd |
dc.source.none.fl_str_mv |
reponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação instacron:RCAAP |
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Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação |
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RCAAP |
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RCAAP |
reponame_str |
Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
collection |
Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
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Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação |
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1799133264389079040 |