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A Human DUB Protein Array for Clarification of Linkage Specificity of Polyubiquitin Chain and Application to Evaluation of Its Inhibitors

Protein ubiquitinations play pivotal roles in many cellular processes, including homeostasis, responses to various stimulations, and progression of diseases. Deubiquitinating enzymes (DUBs) remove ubiquitin molecules from ubiquitinated proteins and cleave the polyubiquitin chain, thus negatively reg...

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Autores principales: Takahashi, Hirotaka, Yamanaka, Satoshi, Kuwada, Shohei, Higaki, Kana, Kido, Kohki, Sato, Yusuke, Fukai, Shuya, Tokunaga, Fuminori, Sawasaki, Tatsuya
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7344921/
https://www.ncbi.nlm.nih.gov/pubmed/32512835
http://dx.doi.org/10.3390/biomedicines8060152
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author Takahashi, Hirotaka
Yamanaka, Satoshi
Kuwada, Shohei
Higaki, Kana
Kido, Kohki
Sato, Yusuke
Fukai, Shuya
Tokunaga, Fuminori
Sawasaki, Tatsuya
author_facet Takahashi, Hirotaka
Yamanaka, Satoshi
Kuwada, Shohei
Higaki, Kana
Kido, Kohki
Sato, Yusuke
Fukai, Shuya
Tokunaga, Fuminori
Sawasaki, Tatsuya
author_sort Takahashi, Hirotaka
collection PubMed
description Protein ubiquitinations play pivotal roles in many cellular processes, including homeostasis, responses to various stimulations, and progression of diseases. Deubiquitinating enzymes (DUBs) remove ubiquitin molecules from ubiquitinated proteins and cleave the polyubiquitin chain, thus negatively regulating numerous ubiquitin-dependent processes. Dysfunctions of many DUBs reportedly cause various diseases; therefore, DUBs are considered as important drug targets, although the biochemical characteristics and cellular functions of many DUBs are still unclear. Here, we established a human DUB protein array to detect the activity and linkage specificity of almost all human DUBs. Using a wheat cell-free protein synthesis system, 88 full-length recombinant human DUB proteins were prepared and termed the DUB array. In vitro DUB assays were performed with all of these recombinant DUBs, using eight linkage types of diubiquitins as substrates. As a result, 80 DUBs in the array showed DUB activities, and their linkage specificities were determined. These 80 DUBs included many biochemically uncharacterized DUBs in the past. In addition, taking advantage of these active DUB proteins, we applied the DUB array to evaluate the selectivities of DUB inhibitors. We successfully developed a high-throughput and semi-quantitative DUB assay based on AlphaScreen technology, and a model study using two commercially available DUB inhibitors revealed individual selectivities to 29 DUBs, as previously reported. In conclusion, the DUB array established here is a powerful tool for biochemical analyses and drug discovery for human DUBs.
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spelling pubmed-73449212020-07-09 A Human DUB Protein Array for Clarification of Linkage Specificity of Polyubiquitin Chain and Application to Evaluation of Its Inhibitors Takahashi, Hirotaka Yamanaka, Satoshi Kuwada, Shohei Higaki, Kana Kido, Kohki Sato, Yusuke Fukai, Shuya Tokunaga, Fuminori Sawasaki, Tatsuya Biomedicines Article Protein ubiquitinations play pivotal roles in many cellular processes, including homeostasis, responses to various stimulations, and progression of diseases. Deubiquitinating enzymes (DUBs) remove ubiquitin molecules from ubiquitinated proteins and cleave the polyubiquitin chain, thus negatively regulating numerous ubiquitin-dependent processes. Dysfunctions of many DUBs reportedly cause various diseases; therefore, DUBs are considered as important drug targets, although the biochemical characteristics and cellular functions of many DUBs are still unclear. Here, we established a human DUB protein array to detect the activity and linkage specificity of almost all human DUBs. Using a wheat cell-free protein synthesis system, 88 full-length recombinant human DUB proteins were prepared and termed the DUB array. In vitro DUB assays were performed with all of these recombinant DUBs, using eight linkage types of diubiquitins as substrates. As a result, 80 DUBs in the array showed DUB activities, and their linkage specificities were determined. These 80 DUBs included many biochemically uncharacterized DUBs in the past. In addition, taking advantage of these active DUB proteins, we applied the DUB array to evaluate the selectivities of DUB inhibitors. We successfully developed a high-throughput and semi-quantitative DUB assay based on AlphaScreen technology, and a model study using two commercially available DUB inhibitors revealed individual selectivities to 29 DUBs, as previously reported. In conclusion, the DUB array established here is a powerful tool for biochemical analyses and drug discovery for human DUBs. MDPI 2020-06-04 /pmc/articles/PMC7344921/ /pubmed/32512835 http://dx.doi.org/10.3390/biomedicines8060152 Text en © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Takahashi, Hirotaka
Yamanaka, Satoshi
Kuwada, Shohei
Higaki, Kana
Kido, Kohki
Sato, Yusuke
Fukai, Shuya
Tokunaga, Fuminori
Sawasaki, Tatsuya
A Human DUB Protein Array for Clarification of Linkage Specificity of Polyubiquitin Chain and Application to Evaluation of Its Inhibitors
title A Human DUB Protein Array for Clarification of Linkage Specificity of Polyubiquitin Chain and Application to Evaluation of Its Inhibitors
title_full A Human DUB Protein Array for Clarification of Linkage Specificity of Polyubiquitin Chain and Application to Evaluation of Its Inhibitors
title_fullStr A Human DUB Protein Array for Clarification of Linkage Specificity of Polyubiquitin Chain and Application to Evaluation of Its Inhibitors
title_full_unstemmed A Human DUB Protein Array for Clarification of Linkage Specificity of Polyubiquitin Chain and Application to Evaluation of Its Inhibitors
title_short A Human DUB Protein Array for Clarification of Linkage Specificity of Polyubiquitin Chain and Application to Evaluation of Its Inhibitors
title_sort human dub protein array for clarification of linkage specificity of polyubiquitin chain and application to evaluation of its inhibitors
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7344921/
https://www.ncbi.nlm.nih.gov/pubmed/32512835
http://dx.doi.org/10.3390/biomedicines8060152
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