A comprehensive platform for the analysis of ubiquitin-like protein modifications using in vivo biotinylation

Post-translational modification by ubiquitin and ubiquitin-like proteins (UbLs) is fundamental for maintaining protein homeostasis. Efficient isolation of UbL conjugates is hampered by multiple factors, including cost and specificity of reagents, removal of UbLs by proteases, distinguishing UbL conj...

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Autores: Lucia, Pirone, Xolalpa, Wendy, Sigurðsson, Jón Otti, Ramírez Sánchez, Juan Manuel, Pérez Fernández, Coralia, González López, Monika, Lafuente Ruiz de Sabando, Ainara, Elortza, Felix, Rodríguez, Manuel S., Mayor Martínez, Ugo, Olsen, Jesper V., Barrio Olano, María Rosa, Sutherland, James D.
Tipo de recurso: artículo
Fecha de publicación:2017
País:España
Institución:Universidad del País Vasco
Repositorio:Addi. Archivo Digital para la Docencia y la Investigación
OAI Identifier:oai:addi.ehu.eus:10810/78547
Acceso en línea:http://hdl.handle.net/10810/78547
Access Level:acceso abierto
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spelling A comprehensive platform for the analysis of ubiquitin-like protein modifications using in vivo biotinylationLucia, PironeXolalpa, WendySigurðsson, Jón OttiRamírez Sánchez, Juan ManuelPérez Fernández, CoraliaGonzález López, MonikaLafuente Ruiz de Sabando, AinaraElortza, FelixRodríguez, Manuel S.Mayor Martínez, UgoOlsen, Jesper V.Barrio Olano, María RosaSutherland, James D.Post-translational modification by ubiquitin and ubiquitin-like proteins (UbLs) is fundamental for maintaining protein homeostasis. Efficient isolation of UbL conjugates is hampered by multiple factors, including cost and specificity of reagents, removal of UbLs by proteases, distinguishing UbL conjugates from interactors, and low quantities of modified substrates. Here we describe bioUbLs, a comprehensive set of tools for studying modifications in Drosophila and mammals, based on multicistronic expression and in vivo biotinylation using the E. coli biotin protein ligase BirA. While the bioUbLs allow rapid validation of UbL conjugation for exogenous or endogenous proteins, the single vector approach can facilitate biotinylation of most proteins of interest. Purification under denaturing conditions inactivates deconjugating enzymes and stringent washes remove UbL interactors and nonspecific background. We demonstrate the utility of the method in Drosophila cells and transgenic flies, identifying an extensive set of putative SUMOylated proteins in both cases. For mammalian cells, we show conjugation and localization for many different UbLs, with the identification of novel potential substrates for UFM1. Ease of use and the flexibility to modify existing vectors will make the bioUbL system a powerful complement to existing strategies for studying this important mode of protein regulation.This work was supported by the Spanish MINECO (BFU2011-25986, BFU2014-52282-P, SAF2013-44782P) and the Consolider Program (BFU2014-57703-REDC), the Departments of Education and Industry of the Basque Government (PI2012/42), and the Bizkaia County and the UPStream consortium (ITN program PITN-GA-2011-290257, EU). This article is based upon work from COST Action (PROTEOSTASIS BM1307), supported by COST (European Cooperation in Science and Technology). Work at Novo Nordisk Foundation Center for Protein Research (CPR) was funded in part by a generous donation from the Novo Nordisk Foundation (Grant number NNF14CC0001).Nature202620262017info:eu-repo/semantics/articleapplication/pdfhttp://hdl.handle.net/10810/78547reponame:Addi. Archivo Digital para la Docencia y la Investigacióninstname:Universidad del País VascoIngléshttps://www.nature.com/articles/srep40756info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by/4.0/This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material.oai:addi.ehu.eus:10810/785472026-06-18T09:23:17Z
dc.title.none.fl_str_mv A comprehensive platform for the analysis of ubiquitin-like protein modifications using in vivo biotinylation
title A comprehensive platform for the analysis of ubiquitin-like protein modifications using in vivo biotinylation
spellingShingle A comprehensive platform for the analysis of ubiquitin-like protein modifications using in vivo biotinylation
Lucia, Pirone
title_short A comprehensive platform for the analysis of ubiquitin-like protein modifications using in vivo biotinylation
title_full A comprehensive platform for the analysis of ubiquitin-like protein modifications using in vivo biotinylation
title_fullStr A comprehensive platform for the analysis of ubiquitin-like protein modifications using in vivo biotinylation
title_full_unstemmed A comprehensive platform for the analysis of ubiquitin-like protein modifications using in vivo biotinylation
title_sort A comprehensive platform for the analysis of ubiquitin-like protein modifications using in vivo biotinylation
dc.creator.none.fl_str_mv Lucia, Pirone
Xolalpa, Wendy
Sigurðsson, Jón Otti
Ramírez Sánchez, Juan Manuel
Pérez Fernández, Coralia
González López, Monika
Lafuente Ruiz de Sabando, Ainara
Elortza, Felix
Rodríguez, Manuel S.
Mayor Martínez, Ugo
Olsen, Jesper V.
Barrio Olano, María Rosa
Sutherland, James D.
author Lucia, Pirone
author_facet Lucia, Pirone
Xolalpa, Wendy
Sigurðsson, Jón Otti
Ramírez Sánchez, Juan Manuel
Pérez Fernández, Coralia
González López, Monika
Lafuente Ruiz de Sabando, Ainara
Elortza, Felix
Rodríguez, Manuel S.
Mayor Martínez, Ugo
Olsen, Jesper V.
Barrio Olano, María Rosa
Sutherland, James D.
author_role author
author2 Xolalpa, Wendy
Sigurðsson, Jón Otti
Ramírez Sánchez, Juan Manuel
Pérez Fernández, Coralia
González López, Monika
Lafuente Ruiz de Sabando, Ainara
Elortza, Felix
Rodríguez, Manuel S.
Mayor Martínez, Ugo
Olsen, Jesper V.
Barrio Olano, María Rosa
Sutherland, James D.
author2_role author
author
author
author
author
author
author
author
author
author
author
author
description Post-translational modification by ubiquitin and ubiquitin-like proteins (UbLs) is fundamental for maintaining protein homeostasis. Efficient isolation of UbL conjugates is hampered by multiple factors, including cost and specificity of reagents, removal of UbLs by proteases, distinguishing UbL conjugates from interactors, and low quantities of modified substrates. Here we describe bioUbLs, a comprehensive set of tools for studying modifications in Drosophila and mammals, based on multicistronic expression and in vivo biotinylation using the E. coli biotin protein ligase BirA. While the bioUbLs allow rapid validation of UbL conjugation for exogenous or endogenous proteins, the single vector approach can facilitate biotinylation of most proteins of interest. Purification under denaturing conditions inactivates deconjugating enzymes and stringent washes remove UbL interactors and nonspecific background. We demonstrate the utility of the method in Drosophila cells and transgenic flies, identifying an extensive set of putative SUMOylated proteins in both cases. For mammalian cells, we show conjugation and localization for many different UbLs, with the identification of novel potential substrates for UFM1. Ease of use and the flexibility to modify existing vectors will make the bioUbL system a powerful complement to existing strategies for studying this important mode of protein regulation.
publishDate 2017
dc.date.none.fl_str_mv 2017
2026
2026
dc.type.none.fl_str_mv info:eu-repo/semantics/article
format article
dc.identifier.none.fl_str_mv http://hdl.handle.net/10810/78547
url http://hdl.handle.net/10810/78547
dc.language.none.fl_str_mv Inglés
language_invalid_str_mv Inglés
dc.relation.none.fl_str_mv https://www.nature.com/articles/srep40756
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
https://creativecommons.org/licenses/by/4.0/
eu_rights_str_mv openAccess
rights_invalid_str_mv https://creativecommons.org/licenses/by/4.0/
dc.format.none.fl_str_mv application/pdf
dc.publisher.none.fl_str_mv Nature
publisher.none.fl_str_mv Nature
dc.source.none.fl_str_mv reponame:Addi. Archivo Digital para la Docencia y la Investigación
instname:Universidad del País Vasco
instname_str Universidad del País Vasco
reponame_str Addi. Archivo Digital para la Docencia y la Investigación
collection Addi. Archivo Digital para la Docencia y la Investigación
repository.name.fl_str_mv
repository.mail.fl_str_mv
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