title: An ultrasensitive system for measuring the USPs and OTULIN activity using Nanoluc as a reporter.
abstract: The deubiquitinating enzymes (DUBs) are a family of isopeptidases responsible for removing the ubiquitin from the ubiquitinated proteins. Identification of inhibitors for DUBs is emerging as an efficient way for discovering potential medicines for disease treatment. However, the high throughput screening (HTS) assay is still not available for all USPs, especially OTULIN. Here, we described a novel steadily quantifiable DUBs assay platform using Nanoluc (Nluc) as reporter. We further demonstrated that the Ub-Nluc assay could be used for HTS of DUBs inhibitors. Moreover, we generated a sensitive system for OTULIN inhibitors screening using Nluc as a reporter. In summary, our data indicate that Ub-Nluc and the improved Ub-Ub-GS-Nluc assay are efficient systems for measuring activities and screening inhibitors of USPs and OTULIN.
authors.affiliation: Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, 500 Dongchuan Road, Shanghai 200241, China.
authors.forename: Yunfei
authors.initials: Y
authors.lastname: Chen
authors.affiliation: Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, 500 Dongchuan Road, Shanghai 200241, China.
authors.forename: Lufan
authors.initials: L
authors.lastname: Wang
authors.affiliation: Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, 500 Dongchuan Road, Shanghai 200241, China.
authors.forename: Xiaomu
authors.initials: X
authors.lastname: Cheng
authors.affiliation: Department of Central Laboratory, Shanghai Tenth People's Hospital of Tongji University, Shanghai 200072, China.
authors.forename: Xin
authors.initials: X
authors.lastname: Ge
authors.affiliation: Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, 500 Dongchuan Road, Shanghai 200241, China; Department of Central Laboratory, Shanghai Tenth People's Hospital of Tongji University, Shanghai 200072, China. Electronic address: pwang@bio.ecnu.edu.cn.
authors.forename: Ping
authors.initials: P
authors.lastname: Wang
mesh_terms: D000465:Algorithms; D000818:Animals; D001671:Biochemistry / Q000379:methods*; D002384:Catalysis; D017930:Genes, Reporter; D049449:Luminescence; D051379:Mice; D010447:Peptide Hydrolases / Q000737:chemistry; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D015203:Reproducibility of Results; D025801:Ubiquitin / Q000737:chemistry*; D064570:Ubiquitin-Specific Proteases / Q000737:chemistry*; D054875:Ubiquitination
chemical_list: D011993:Recombinant Fusion Proteins; D025801:Ubiquitin; D010447:Peptide Hydrolases; D064570:Ubiquitin-Specific Proteases
keywords: Deubiquitinating enzymes; Nanoluc; OTULIN; PR619; Ubiquitin