A fluorescence based sensor assay that monitors general protein aggregation in human cells
Autor(a) principal: | |
---|---|
Data de Publicação: | 2018 |
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/10773/27440 |
Resumo: | Protein conformational disorders are characterized by disruption of protein folding and toxic accumulation of protein aggregates. Here we describe a sensitive and simple method to follow and monitor general protein aggregation in human cells. Heat shock protein 27 (HSP27) is an oligomeric small heat shock protein that binds and keeps unfolded proteins in a folding competent state. This high specificity of HSP27 for aggregated proteins can be explored to monitor aggregation in living cells by fusing it to a fluorescent protein as Green Fluorescent Protein (GFP). We have constructed a HeLa stable cell line expressing a HSP27:GFP chimeric reporter protein and after validation, this stable cell line is exposed to different agents that interfere with proteostasis, namely Arsenite, MG132, and Aβ-peptide. Exposure to proteome destabilizers lead to re-localization of HSP27:GFP fluorescence to foci, confirming that our reporter system is functional and can be used to detect and follow protein aggregation in living cells. This reporter is a valuable tool to setup wide-genetic screens to identify genes and pathways involved in protein misfolding and aggregation. |
id |
RCAP_5099f34c82d02debfc88b05358124556 |
---|---|
oai_identifier_str |
oai:ria.ua.pt:10773/27440 |
network_acronym_str |
RCAP |
network_name_str |
Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
repository_id_str |
7160 |
spelling |
A fluorescence based sensor assay that monitors general protein aggregation in human cellsFluorescence sensor assayGFP Green Fluorescent ProteinsHSP27 Heat-Shock ProteinsHuman cellsProtein aggregationProtein foldingProteomeRecombinant proteinsAmyloid beta-peptidesProtein conformational disorders are characterized by disruption of protein folding and toxic accumulation of protein aggregates. Here we describe a sensitive and simple method to follow and monitor general protein aggregation in human cells. Heat shock protein 27 (HSP27) is an oligomeric small heat shock protein that binds and keeps unfolded proteins in a folding competent state. This high specificity of HSP27 for aggregated proteins can be explored to monitor aggregation in living cells by fusing it to a fluorescent protein as Green Fluorescent Protein (GFP). We have constructed a HeLa stable cell line expressing a HSP27:GFP chimeric reporter protein and after validation, this stable cell line is exposed to different agents that interfere with proteostasis, namely Arsenite, MG132, and Aβ-peptide. Exposure to proteome destabilizers lead to re-localization of HSP27:GFP fluorescence to foci, confirming that our reporter system is functional and can be used to detect and follow protein aggregation in living cells. This reporter is a valuable tool to setup wide-genetic screens to identify genes and pathways involved in protein misfolding and aggregation.Wiley2020-01-31T14:53:58Z2018-04-01T00:00:00Z2018-04info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articleapplication/pdfhttp://hdl.handle.net/10773/27440eng1860-676810.1002/biot.201700676Pereira, MarisaTomé, DiogoDomingues, Ana S.Varanda, Ana S.Paulo, CristianaSantos, Manuel A. S.Soares, Ana R.info: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:RCAAP2024-02-22T11:52:05Zoai:ria.ua.pt:10773/27440Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireopendoar:71602024-03-20T02:59:47.744482Repositório 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 |
A fluorescence based sensor assay that monitors general protein aggregation in human cells |
title |
A fluorescence based sensor assay that monitors general protein aggregation in human cells |
spellingShingle |
A fluorescence based sensor assay that monitors general protein aggregation in human cells Pereira, Marisa Fluorescence sensor assay GFP Green Fluorescent Proteins HSP27 Heat-Shock Proteins Human cells Protein aggregation Protein folding Proteome Recombinant proteins Amyloid beta-peptides |
title_short |
A fluorescence based sensor assay that monitors general protein aggregation in human cells |
title_full |
A fluorescence based sensor assay that monitors general protein aggregation in human cells |
title_fullStr |
A fluorescence based sensor assay that monitors general protein aggregation in human cells |
title_full_unstemmed |
A fluorescence based sensor assay that monitors general protein aggregation in human cells |
title_sort |
A fluorescence based sensor assay that monitors general protein aggregation in human cells |
author |
Pereira, Marisa |
author_facet |
Pereira, Marisa Tomé, Diogo Domingues, Ana S. Varanda, Ana S. Paulo, Cristiana Santos, Manuel A. S. Soares, Ana R. |
author_role |
author |
author2 |
Tomé, Diogo Domingues, Ana S. Varanda, Ana S. Paulo, Cristiana Santos, Manuel A. S. Soares, Ana R. |
author2_role |
author author author author author author |
dc.contributor.author.fl_str_mv |
Pereira, Marisa Tomé, Diogo Domingues, Ana S. Varanda, Ana S. Paulo, Cristiana Santos, Manuel A. S. Soares, Ana R. |
dc.subject.por.fl_str_mv |
Fluorescence sensor assay GFP Green Fluorescent Proteins HSP27 Heat-Shock Proteins Human cells Protein aggregation Protein folding Proteome Recombinant proteins Amyloid beta-peptides |
topic |
Fluorescence sensor assay GFP Green Fluorescent Proteins HSP27 Heat-Shock Proteins Human cells Protein aggregation Protein folding Proteome Recombinant proteins Amyloid beta-peptides |
description |
Protein conformational disorders are characterized by disruption of protein folding and toxic accumulation of protein aggregates. Here we describe a sensitive and simple method to follow and monitor general protein aggregation in human cells. Heat shock protein 27 (HSP27) is an oligomeric small heat shock protein that binds and keeps unfolded proteins in a folding competent state. This high specificity of HSP27 for aggregated proteins can be explored to monitor aggregation in living cells by fusing it to a fluorescent protein as Green Fluorescent Protein (GFP). We have constructed a HeLa stable cell line expressing a HSP27:GFP chimeric reporter protein and after validation, this stable cell line is exposed to different agents that interfere with proteostasis, namely Arsenite, MG132, and Aβ-peptide. Exposure to proteome destabilizers lead to re-localization of HSP27:GFP fluorescence to foci, confirming that our reporter system is functional and can be used to detect and follow protein aggregation in living cells. This reporter is a valuable tool to setup wide-genetic screens to identify genes and pathways involved in protein misfolding and aggregation. |
publishDate |
2018 |
dc.date.none.fl_str_mv |
2018-04-01T00:00:00Z 2018-04 2020-01-31T14:53:58Z |
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/10773/27440 |
url |
http://hdl.handle.net/10773/27440 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
1860-6768 10.1002/biot.201700676 |
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 |
Wiley |
publisher.none.fl_str_mv |
Wiley |
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 |
instname_str |
Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação |
instacron_str |
RCAAP |
institution |
RCAAP |
reponame_str |
Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
collection |
Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
repository.name.fl_str_mv |
Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação |
repository.mail.fl_str_mv |
|
_version_ |
1799137652621967360 |