title: A fluorescence assay for elucidating the substrate specificities of deubiquitinating enzymes.
abstract: Ubiquitin C-terminal hydrolases (UCHs) are a representative family of deubiquitinating enzymes (DUBs), which specifically cleave ubiquitin (Ub) chains or extensions. Here we present a convenient method for characterizing the substrate specificities of various UCHs by fluorescently mutated Ub-fusion proteins (Ub(F45W)-Xaa) and di-ubiquitin chains (Ub(F45W)-diUb). After removal of the intact substrate by Ni(2+)-NTA affinity, the enzymatic activities of UCHs were quantitatively determined by recording fluorescence of the Ub(F45W) product. The results show that three UCHs, i.e. UCH-L1, UCH-L3 and UCH37/UCH-L5, are distinct in their substrate specificities for the Ub-fusions and diUb chains. This assay method may also be applied to study the enzymatic activities and substrate specificities of other DUBs.
authors.affiliation: State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.
authors.forename: Si-Tao
authors.initials: ST
authors.lastname: Yin
authors.forename: Hao
authors.initials: H
authors.lastname: Huang
authors.forename: Yu-Hang
authors.initials: YH
authors.lastname: Zhang
authors.forename: Zi-Ren
authors.initials: ZR
authors.lastname: Zhou
authors.forename: Ai-Xin
authors.initials: AX
authors.lastname: Song
authors.forename: Fa-Shui
authors.initials: FS
authors.lastname: Hong
authors.forename: Hong-Yu
authors.initials: HY
authors.lastname: Hu
mesh_terms: D002268:Carboxypeptidases / Q000737:chemistry*; D003546:Cysteine Endopeptidases / Q000737:chemistry*; D006801:Humans; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013379:Substrate Specificity; D043222:Ubiquitin Thiolesterase / Q000737:chemistry*
chemical_list: C482860:UCHL1 protein, human; D002268:Carboxypeptidases; C509806:UCHL3 protein, human; C421245:UCHL5 protein, human; D043222:Ubiquitin Thiolesterase; D003546:Cysteine Endopeptidases