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Open AccessArticle

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

1
Proteo-Science Center, Ehime University, Matsuyama 790-8577, Japan
2
Center for Research on Green Sustainable Chemistry, Tottori University, Tottori 680-8552, Japan
3
Department of Chemistry, Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan
4
Department of Pathobiochemistry, Graduate School of Medicine, Osaka City University, Osaka 545-8585, Japan
*
Authors to whom correspondence should be addressed.
Biomedicines 2020, 8(6), 152; https://doi.org/10.3390/biomedicines8060152
Received: 28 April 2020 / Revised: 2 June 2020 / Accepted: 3 June 2020 / Published: 4 June 2020
(This article belongs to the Section Tumor Cell Biology)
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. View Full-Text
Keywords: deubiquitinating enzyme; polyubiquitin chain linkage; DUB inhibitor; wheat cell-free system; protein array technology deubiquitinating enzyme; polyubiquitin chain linkage; DUB inhibitor; wheat cell-free system; protein array technology
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MDPI and ACS Style

Takahashi, H.; Yamanaka, S.; Kuwada, S.; Higaki, K.; Kido, K.; Sato, Y.; Fukai, S.; Tokunaga, F.; Sawasaki, T. A Human DUB Protein Array for Clarification of Linkage Specificity of Polyubiquitin Chain and Application to Evaluation of Its Inhibitors. Biomedicines 2020, 8, 152. https://doi.org/10.3390/biomedicines8060152

AMA Style

Takahashi H, Yamanaka S, Kuwada S, Higaki K, Kido K, Sato Y, Fukai S, Tokunaga F, Sawasaki T. A Human DUB Protein Array for Clarification of Linkage Specificity of Polyubiquitin Chain and Application to Evaluation of Its Inhibitors. Biomedicines. 2020; 8(6):152. https://doi.org/10.3390/biomedicines8060152

Chicago/Turabian Style

Takahashi, Hirotaka; Yamanaka, Satoshi; Kuwada, Shohei; Higaki, Kana; Kido, Kohki; Sato, Yusuke; Fukai, Shuya; Tokunaga, Fuminori; Sawasaki, Tatsuya. 2020. "A Human DUB Protein Array for Clarification of Linkage Specificity of Polyubiquitin Chain and Application to Evaluation of Its Inhibitors" Biomedicines 8, no. 6: 152. https://doi.org/10.3390/biomedicines8060152

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