A New Fluorescence-Based Method Identifies Protein Phosphatases Regulating Lipid Droplet Metabolism

Detalhes bibliográficos
Autor(a) principal: Bozaquel-Morais, Bruno L.
Data de Publicação: 2010
Outros Autores: Madeira, Juliana B, Monteiro, Clarissa M. Maya, Masuda, Claudio A., Lomeli, Mónica Montero
Tipo de documento: Artigo
Idioma: eng
Título da fonte: Repositório Institucional da FIOCRUZ (ARCA)
Texto Completo: https://www.arca.fiocruz.br/handle/icict/30453
Resumo: Universidade Federal do Rio de Janeiro. Instituto de Bioquímica Médica. Programa de Biologia Molecular e Biotecnologia. Rio de Janeiro, RJ, Brasil.
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spelling Bozaquel-Morais, Bruno L.Madeira, Juliana BMonteiro, Clarissa M. MayaMasuda, Claudio A.Lomeli, Mónica Montero2018-12-09T18:44:21Z2018-12-09T18:44:21Z2010BOZAQUEL-MORAIS, Bruno L. et al A New Fluorescence-Based Method Identifies Protein Phosphatases Regulating Lipid Droplet Metabolism. Plos One, v.5, n.10, e13692, 14p, Oct. 2010.1932-6203https://www.arca.fiocruz.br/handle/icict/30453engPublic Library of ScienceMétodo Baseado Em FluorescênciaFosfatasesProteínaGotas LipídicasMetabolismoLipid DropletMetabolismFluorescence-Based MethodPhosphatasesproteinA New Fluorescence-Based Method Identifies Protein Phosphatases Regulating Lipid Droplet Metabolisminfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articleUniversidade Federal do Rio de Janeiro. Instituto de Bioquímica Médica. Programa de Biologia Molecular e Biotecnologia. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Instituto de Bioquímica Médica. Programa de Biologia Molecular e Biotecnologia. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Imunofarmacologia. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Instituto de Bioquímica Médica. Programa de Biologia Molecular e Biotecnologia. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Instituto de Bioquímica Médica. Programa de Biologia Molecular e Biotecnologia. Rio de Janeiro, RJ, Brasil.In virtually every cell, neutral lipids are stored in cytoplasmic structures called lipid droplets (LDs) and also referred to as lipid bodies or lipid particles. We developed a rapid high-throughput assay based on the recovery of quenched BODIPYfluorescence that allows to quantify lipid droplets. The method was validated by monitoring lipid droplet turnover during growth of a yeast culture and by screening a group of strains deleted in genes known to be involved in lipid metabolism. In both tests, the fluorimetric assay showed high sensitivity and good agreement with previously reported data using microscopy. We used this method for high-throughput identification of protein phosphatases involved in lipid droplet metabolism. From 65 yeast knockout strains encoding protein phosphatases and its regulatory subunits, 13 strains revealed to have abnormal levels of lipid droplets, 10 of them having high lipid droplet content. Strains deleted for type I protein phosphatases and related regulators (ppz2, gac1, bni4), type 2A phosphatase and its related regulator (pph21 and sap185), type 2C protein phosphatases (ptc1, ptc4, ptc7) and dual phosphatases (pps1, msg5) were catalogued as high-lipid droplet content strains. Only reg1, a targeting subunit of the type 1 phosphatase Glc7p, and members of the nutrient-sensitive TOR pathway (sit4 and the regulatory subunit sap190) were catalogued as low-lipid droplet content strains, which were studied further. We show that Snf1, the homologue of the mammalian AMP-activated kinase, is constitutively phosphorylated (hyperactive) in sit4 and sap190 strains leading to a reduction of acetyl-CoA carboxylase activity. In conclusion, our fast and highly sensitive method permitted us to catalogue protein phosphatases involved in the regulation of LD metabolism and present evidence indicating that the TOR pathway and the SNF1/AMPK pathway are connected through the Sit4p-Sap190p pair in the control of lipid droplet biogenesis.info:eu-repo/semantics/openAccessreponame:Repositório Institucional da FIOCRUZ (ARCA)instname:Fundação Oswaldo Cruz (FIOCRUZ)instacron:FIOCRUZLICENSElicense.txtlicense.txttext/plain; charset=utf-82991https://www.arca.fiocruz.br/bitstream/icict/30453/1/license.txt5a560609d32a3863062d77ff32785d58MD51ORIGINALclarissamaia_monteiro_etal_IOC_2010.pdfclarissamaia_monteiro_etal_IOC_2010.pdfapplication/pdf795973https://www.arca.fiocruz.br/bitstream/icict/30453/2/clarissamaia_monteiro_etal_IOC_2010.pdf1080cd4b20c451400a0b688cda53906cMD52TEXTclarissamaia_monteiro_etal_IOC_2010.pdf.txtclarissamaia_monteiro_etal_IOC_2010.pdf.txtExtracted 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dc.title.pt_BR.fl_str_mv A New Fluorescence-Based Method Identifies Protein Phosphatases Regulating Lipid Droplet Metabolism
title A New Fluorescence-Based Method Identifies Protein Phosphatases Regulating Lipid Droplet Metabolism
spellingShingle A New Fluorescence-Based Method Identifies Protein Phosphatases Regulating Lipid Droplet Metabolism
Bozaquel-Morais, Bruno L.
Método Baseado Em Fluorescência
Fosfatases
Proteína
Gotas Lipídicas
Metabolismo
Lipid Droplet
Metabolism
Fluorescence-Based Method
Phosphatases
protein
title_short A New Fluorescence-Based Method Identifies Protein Phosphatases Regulating Lipid Droplet Metabolism
title_full A New Fluorescence-Based Method Identifies Protein Phosphatases Regulating Lipid Droplet Metabolism
title_fullStr A New Fluorescence-Based Method Identifies Protein Phosphatases Regulating Lipid Droplet Metabolism
title_full_unstemmed A New Fluorescence-Based Method Identifies Protein Phosphatases Regulating Lipid Droplet Metabolism
title_sort A New Fluorescence-Based Method Identifies Protein Phosphatases Regulating Lipid Droplet Metabolism
author Bozaquel-Morais, Bruno L.
author_facet Bozaquel-Morais, Bruno L.
Madeira, Juliana B
Monteiro, Clarissa M. Maya
Masuda, Claudio A.
Lomeli, Mónica Montero
author_role author
author2 Madeira, Juliana B
Monteiro, Clarissa M. Maya
Masuda, Claudio A.
Lomeli, Mónica Montero
author2_role author
author
author
author
dc.contributor.author.fl_str_mv Bozaquel-Morais, Bruno L.
Madeira, Juliana B
Monteiro, Clarissa M. Maya
Masuda, Claudio A.
Lomeli, Mónica Montero
dc.subject.other.pt_BR.fl_str_mv Método Baseado Em Fluorescência
Fosfatases
Proteína
Gotas Lipídicas
Metabolismo
topic Método Baseado Em Fluorescência
Fosfatases
Proteína
Gotas Lipídicas
Metabolismo
Lipid Droplet
Metabolism
Fluorescence-Based Method
Phosphatases
protein
dc.subject.en.pt_BR.fl_str_mv Lipid Droplet
Metabolism
Fluorescence-Based Method
Phosphatases
protein
description Universidade Federal do Rio de Janeiro. Instituto de Bioquímica Médica. Programa de Biologia Molecular e Biotecnologia. Rio de Janeiro, RJ, Brasil.
publishDate 2010
dc.date.issued.fl_str_mv 2010
dc.date.accessioned.fl_str_mv 2018-12-09T18:44:21Z
dc.date.available.fl_str_mv 2018-12-09T18:44:21Z
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.citation.fl_str_mv BOZAQUEL-MORAIS, Bruno L. et al A New Fluorescence-Based Method Identifies Protein Phosphatases Regulating Lipid Droplet Metabolism. Plos One, v.5, n.10, e13692, 14p, Oct. 2010.
dc.identifier.uri.fl_str_mv https://www.arca.fiocruz.br/handle/icict/30453
dc.identifier.issn.pt_BR.fl_str_mv 1932-6203
identifier_str_mv BOZAQUEL-MORAIS, Bruno L. et al A New Fluorescence-Based Method Identifies Protein Phosphatases Regulating Lipid Droplet Metabolism. Plos One, v.5, n.10, e13692, 14p, Oct. 2010.
1932-6203
url https://www.arca.fiocruz.br/handle/icict/30453
dc.language.iso.fl_str_mv eng
language eng
dc.rights.driver.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.publisher.none.fl_str_mv Public Library of Science
publisher.none.fl_str_mv Public Library of Science
dc.source.none.fl_str_mv reponame:Repositório Institucional da FIOCRUZ (ARCA)
instname:Fundação Oswaldo Cruz (FIOCRUZ)
instacron:FIOCRUZ
instname_str Fundação Oswaldo Cruz (FIOCRUZ)
instacron_str FIOCRUZ
institution FIOCRUZ
reponame_str Repositório Institucional da FIOCRUZ (ARCA)
collection Repositório Institucional da FIOCRUZ (ARCA)
bitstream.url.fl_str_mv https://www.arca.fiocruz.br/bitstream/icict/30453/1/license.txt
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https://www.arca.fiocruz.br/bitstream/icict/30453/3/clarissamaia_monteiro_etal_IOC_2010.pdf.txt
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repository.mail.fl_str_mv repositorio.arca@fiocruz.br
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