Expression, purification and enzymatic characterization of a recombinant human ubiquitin-specific protease 47
| العنوان: | Expression, purification and enzymatic characterization of a recombinant human ubiquitin-specific protease 47 |
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| المؤلفون: | Akira Hattori, Jinhua Piao, Eiko Moriyoshi, Aika Tashiro, Minako Nishikawa, Yutaka Aoki, Hideaki Kakeya |
| المصدر: | Journal of Biochemistry. :mvv063 |
| بيانات النشر: | Oxford University Press (OUP), 2015. |
| سنة النشر: | 2015 |
| مصطلحات موضوعية: | medicine.medical_treatment, Biochemistry, Substrate Specificity, law.invention, Ubiquitin-Specific Peptidase 7, Ubiquitin, law, medicine, Humans, Enzyme Inhibitors, Ubiquitins, Molecular Biology, Polyacrylamide gel electrophoresis, Gel electrophoresis, Protease, Molecular mass, biology, Chemistry, Ubiquitination, Proteolytic enzymes, General Medicine, Molecular biology, Recombinant Proteins, Kinetics, Zinc, Amino Acid Substitution, Ethylmaleimide, Recombinant DNA, biology.protein, Electrophoresis, Polyacrylamide Gel, Ubiquitin-Specific Proteases, Baculoviridae, Ubiquitin Thiolesterase, Cysteine |
| الوصف: | 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. |
| تدمد: | 1756-2651 0021-924X |
| URL الوصول: | https://explore.openaire.eu/search/publication?articleId=doi_dedup___::e8588a49469037a9d024e228e36f1c45 https://doi.org/10.1093/jb/mvv063 |
| رقم الأكسشن: | edsair.doi.dedup.....e8588a49469037a9d024e228e36f1c45 |
| قاعدة البيانات: | OpenAIRE |
| تدمد: | 17562651 0021924X |
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