title: Synthesis and characterization of fluorescent ubiquitin derivatives as highly sensitive substrates for the deubiquitinating enzymes UCH-L3 and USP-2.
abstract: Deubiquitinating enzymes (DUBs) catalyze the removal of attached ubiquitin molecules from amino groups of target proteins. The large family of DUBs plays an important role in the regulation of the intracellular homeostasis of different proteins and influences therefore key events such as cell division, apoptosis, etc. The DUB family members UCH-L3 and USP2 are believed to inhibit the degradation of various tumor-growth-promoting proteins by removing the trigger for degradation. Inhibitors of these enzymes should therefore lead to enhanced degradation of oncoproteins and may thus stop tumor growth. To develop an enzymatic assay for the search of UCH-L3 and USP2 inhibitors, C-terminally labeled ubiquitin substrates were enzymatically synthesized. We have used the ubiquitin-activating enzyme E1 and one of the ubiquitin-conjugating enzymes E2 to attach a fluorescent lysine derivative to the C terminus of ubiquitin. Since only the epsilon-NH(2) group of the lysine derivatives was free and reactive, the conjugates closely mimic the isopeptide bond between the ubiquitin and the lysine side chains of the targeted proteins. Various substrates were synthesized by this approach and characterized enzymatically with the two DUBs. The variant consisting of the fusion protein between the large N-terminal NusA tag and the ubiquitin which was modified with alpha-NH(2)-tetramethylrhodamin-lysine, was found to give the highest dynamic range in a fluorescence polarization readout. Therefore we have chosen this substrate for the development of a miniaturized, fluorescence-polarization-based high-throughput screening assay.
authors.affiliation: Discovery Technologies, Novartis Institutes for Biomedical Research, CH-4058 Basel, Switzerland.
authors.forename: Aline
authors.initials: A
authors.lastname: Tirat
authors.forename: Alain
authors.initials: A
authors.lastname: Schilb
authors.forename: Virginie
authors.initials: V
authors.lastname: Riou
authors.forename: Lukas
authors.initials: L
authors.lastname: Leder
authors.forename: Bernd
authors.initials: B
authors.lastname: Gerhartz
authors.forename: Johann
authors.initials: J
authors.lastname: Zimmermann
authors.forename: Susanne
authors.initials: S
authors.lastname: Worpenberg
authors.forename: Ulf
authors.initials: U
authors.lastname: Eidhoff
authors.forename: Felix
authors.initials: F
authors.lastname: Freuler
authors.forename: Thomas
authors.initials: T
authors.lastname: Stettler
authors.forename: Lorenz
authors.initials: L
authors.lastname: Mayr
authors.forename: Johannes
authors.initials: J
authors.lastname: Ottl
authors.forename: Beate
authors.initials: B
authors.lastname: Leuenberger
authors.forename: Ireos
authors.initials: I
authors.lastname: Filipuzzi
mesh_terms: D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000737:chemistry / Q000378:metabolism; D005456:Fluorescent Dyes / Q000138:chemical synthesis* / Q000737:chemistry; D007700:Kinetics; D008239:Lysine / Q000737:chemistry / Q000378:metabolism; D012235:Rhodamines / Q000737:chemistry / Q000378:metabolism; D013997:Time Factors; D025801:Ubiquitin / Q000031:analogs & derivatives* / Q000138:chemical synthesis* / Q000737:chemistry; D043222:Ubiquitin Thiolesterase / Q000737:chemistry / Q000378:metabolism
chemical_list: C114557:5-carboxytetramethylrhodamine succinimidyl ester; D005456:Fluorescent Dyes; D012235:Rhodamines; C482860:UCHL1 protein, human; D025801:Ubiquitin; D010450:Endopeptidases; C108665:USP2 protein, human; D043222:Ubiquitin Thiolesterase; D008239:Lysine