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Non-hydrolyzable Diubiquitin Probes Reveal Linkage-Specific Reactivity of Deubiquitylating Enzymes Mediated by S2 Pockets
Ubiquitin chains are important post-translational modifications that control a large number of cellular processes. Chains can be formed via different linkages, which determines the type of signal they convey. Deubiquitylating enzymes (DUBs) regulate ubiquitylation status by trimming or removing chai...
Autores principales: | , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Cell Press
2016
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4850247/ https://www.ncbi.nlm.nih.gov/pubmed/27066941 http://dx.doi.org/10.1016/j.chembiol.2016.03.009 |
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author | Flierman, Dennis van der Heden van Noort, Gerbrand J. Ekkebus, Reggy Geurink, Paul P. Mevissen, Tycho E.T. Hospenthal, Manuela K. Komander, David Ovaa, Huib |
author_facet | Flierman, Dennis van der Heden van Noort, Gerbrand J. Ekkebus, Reggy Geurink, Paul P. Mevissen, Tycho E.T. Hospenthal, Manuela K. Komander, David Ovaa, Huib |
author_sort | Flierman, Dennis |
collection | PubMed |
description | Ubiquitin chains are important post-translational modifications that control a large number of cellular processes. Chains can be formed via different linkages, which determines the type of signal they convey. Deubiquitylating enzymes (DUBs) regulate ubiquitylation status by trimming or removing chains from attached proteins. DUBs can contain several ubiquitin-binding pockets, which confer specificity toward differently linked chains. Most tools for monitoring DUB specificity target binding pockets on opposing sides of the active site; however, some DUBs contain additional pockets. Therefore, reagents targeting additional pockets are essential to fully understand linkage specificity. We report the development of active site-directed probes and fluorogenic substrates, based on non-hydrolyzable diubiquitin, that are equipped with a C-terminal warhead or a fluorogenic activity reporter moiety. We demonstrate that various DUBs in lysates display differential reactivity toward differently linked diubiquitin probes, as exemplified by the proteasome-associated DUB USP14. In addition, OTUD2 and OTUD3 show remarkable linkage-specific reactivity with our diubiquitin-based reagents. |
format | Online Article Text |
id | pubmed-4850247 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2016 |
publisher | Cell Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-48502472016-05-06 Non-hydrolyzable Diubiquitin Probes Reveal Linkage-Specific Reactivity of Deubiquitylating Enzymes Mediated by S2 Pockets Flierman, Dennis van der Heden van Noort, Gerbrand J. Ekkebus, Reggy Geurink, Paul P. Mevissen, Tycho E.T. Hospenthal, Manuela K. Komander, David Ovaa, Huib Cell Chem Biol Article Ubiquitin chains are important post-translational modifications that control a large number of cellular processes. Chains can be formed via different linkages, which determines the type of signal they convey. Deubiquitylating enzymes (DUBs) regulate ubiquitylation status by trimming or removing chains from attached proteins. DUBs can contain several ubiquitin-binding pockets, which confer specificity toward differently linked chains. Most tools for monitoring DUB specificity target binding pockets on opposing sides of the active site; however, some DUBs contain additional pockets. Therefore, reagents targeting additional pockets are essential to fully understand linkage specificity. We report the development of active site-directed probes and fluorogenic substrates, based on non-hydrolyzable diubiquitin, that are equipped with a C-terminal warhead or a fluorogenic activity reporter moiety. We demonstrate that various DUBs in lysates display differential reactivity toward differently linked diubiquitin probes, as exemplified by the proteasome-associated DUB USP14. In addition, OTUD2 and OTUD3 show remarkable linkage-specific reactivity with our diubiquitin-based reagents. Cell Press 2016-04-21 /pmc/articles/PMC4850247/ /pubmed/27066941 http://dx.doi.org/10.1016/j.chembiol.2016.03.009 Text en © 2016 The Authors http://creativecommons.org/licenses/by/4.0/ This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Flierman, Dennis van der Heden van Noort, Gerbrand J. Ekkebus, Reggy Geurink, Paul P. Mevissen, Tycho E.T. Hospenthal, Manuela K. Komander, David Ovaa, Huib Non-hydrolyzable Diubiquitin Probes Reveal Linkage-Specific Reactivity of Deubiquitylating Enzymes Mediated by S2 Pockets |
title | Non-hydrolyzable Diubiquitin Probes Reveal Linkage-Specific Reactivity of Deubiquitylating Enzymes Mediated by S2 Pockets |
title_full | Non-hydrolyzable Diubiquitin Probes Reveal Linkage-Specific Reactivity of Deubiquitylating Enzymes Mediated by S2 Pockets |
title_fullStr | Non-hydrolyzable Diubiquitin Probes Reveal Linkage-Specific Reactivity of Deubiquitylating Enzymes Mediated by S2 Pockets |
title_full_unstemmed | Non-hydrolyzable Diubiquitin Probes Reveal Linkage-Specific Reactivity of Deubiquitylating Enzymes Mediated by S2 Pockets |
title_short | Non-hydrolyzable Diubiquitin Probes Reveal Linkage-Specific Reactivity of Deubiquitylating Enzymes Mediated by S2 Pockets |
title_sort | non-hydrolyzable diubiquitin probes reveal linkage-specific reactivity of deubiquitylating enzymes mediated by s2 pockets |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4850247/ https://www.ncbi.nlm.nih.gov/pubmed/27066941 http://dx.doi.org/10.1016/j.chembiol.2016.03.009 |
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