title: Expression, purification and enzymatic characterization of a recombinant human ubiquitin-specific protease 47.
abstract: In this study, the physicochemical and enzymatic properties of recombinant human ubiquitin (Ub)-specific protease (USP) 47, a novel member of the C19 family of de-ubiquitinating enzymes (DUB), were characterized for the first time. Recombinant human USP47 was expressed in a baculovirus expression system and purified to homogeneity. The purified protein was shown to be a monomeric protein with a molecular mass of ∼146 kDa on sodium dodecyl sulphate-polyacrylamide gel electrophoresis. USP47 released Ub from Ub-aminoacyl-4-metheylcoumaryl-7-amide and Ub-tagged granzyme B. The substitution of the potential nucleophile Cys109 with Ser severely abrogated the Ub-releasing activity of USP47, indicating that USP47 is indeed a cysteine DUB. An assay using Ub dimer substrates showed that the enzyme cleaved a variety of isopeptide bonds between 2 Ub molecules, including the Lys48- and Lys63-linked isopeptide bonds. USP47 also released a Ub moiety from Lys48- and Lys63-linked polyUb chains. Of the inhibitors tested, N-ethylmaleimide, Zn ion and Ub aldehyde revealed a dose-dependent inhibition of USP47. In this study, clear differences in the enzymatic properties between USP47 and USP7 (the most closely related proteins among DUBs) were also found. Therefore, our results suggest that USP47 may play distinct roles in Ub-mediated cellular processes via DUB activity.
authors.affiliation: Department of System Chemotherapy and Molecular Sciences, Division of Bioinformatics and Chemical Genomics, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo, Kyoto 606-8501, Japan.
authors.forename: Jinhua
authors.initials: J
authors.lastname: Piao
authors.affiliation: Department of System Chemotherapy and Molecular Sciences, Division of Bioinformatics and Chemical Genomics, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo, Kyoto 606-8501, Japan.
authors.forename: Aika
authors.initials: A
authors.lastname: Tashiro
authors.affiliation: Department of System Chemotherapy and Molecular Sciences, Division of Bioinformatics and Chemical Genomics, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo, Kyoto 606-8501, Japan.
authors.forename: Minako
authors.initials: M
authors.lastname: Nishikawa
authors.affiliation: Department of System Chemotherapy and Molecular Sciences, Division of Bioinformatics and Chemical Genomics, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo, Kyoto 606-8501, Japan.
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Aoki
authors.affiliation: Department of System Chemotherapy and Molecular Sciences, Division of Bioinformatics and Chemical Genomics, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo, Kyoto 606-8501, Japan.
authors.forename: Eiko
authors.initials: E
authors.lastname: Moriyoshi
authors.affiliation: Department of System Chemotherapy and Molecular Sciences, Division of Bioinformatics and Chemical Genomics, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo, Kyoto 606-8501, Japan ahattori@pharm.kyoto-u.ac.jp.
authors.forename: Akira
authors.initials: A
authors.lastname: Hattori
authors.affiliation: Department of System Chemotherapy and Molecular Sciences, Division of Bioinformatics and Chemical Genomics, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo, Kyoto 606-8501, Japan ahattori@pharm.kyoto-u.ac.jp.
authors.forename: Hideaki
authors.initials: H
authors.lastname: Kakeya
mesh_terms: D019943:Amino Acid Substitution; D016367:Baculoviridae; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D013379:Substrate Specificity; D025801:Ubiquitin / Q000378:metabolism*; D043222:Ubiquitin Thiolesterase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification; D000076084:Ubiquitin-Specific Peptidase 7; D064570:Ubiquitin-Specific Proteases; D054875:Ubiquitination; D014452:Ubiquitins / Q000494:pharmacology; D015032:Zinc / Q000494:pharmacology
chemical_list: D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; C557800:USP47 protein, human; D025801:Ubiquitin; D014452:Ubiquitins; C051927:ubiquitin-aldehyde; C464569:USP7 protein, human; D043222:Ubiquitin Thiolesterase; D000076084:Ubiquitin-Specific Peptidase 7; D064570:Ubiquitin-Specific Proteases; D015032:Zinc; D005033:Ethylmaleimide
keywords: C19 cysteine peptidase family; de-ubiquitinating enzyme; ubiquitin; ubiquitin-specific protease