title: Profiling the Activity of Deubiquitinating Enzymes Using Chemically Synthesized Ubiquitin-Based Probes.
abstract: Deubiquitinating enzymes (DUBs) are of interest as potential new targets for pharmacological intervention. Active-site-directed probes can be used for the accurate profiling of DUB activity as well as the identification of DUBs and DUB inhibitor selectivity. Previously, active-site directed DUB probes have been obtained using intein-based methods that have inherent limitations. Total chemical synthesis of ubiquitin allows for easy incorporation of different tags, such as fluorescent reporters, affinity tags, and cleavable linkers. Here, we describe the total chemical synthesis of a fluorescent active-site directed DUB probe, which facilitates fast, in-gel detection of active DUBs and circumvents the use of Western blot analysis. In addition, an in-gel activity-based DUB profiling assay is described in detail, in which the fluorescent DUB probe is used to visualize active DUBs in cell lysates. Finally, an inhibition assay is described in which the fluorescent probe is used to determine the specificity and potency of a small molecule DUB inhibitor.
authors: Department of Chemical Immunology, Leiden University Medical Center, Albinusdreef 2, 2333, ZA, Leiden, The Netherlands.|Division of Cell Biology II, Netherlands Cancer Institute, Plesmanlaan 121, 1066, CX, Amsterdam, The Netherlands.
authors: Yves
authors: Y
authors: Leestemaker
authors: Division of Cell Biology II, Netherlands Cancer Institute, Plesmanlaan 121, 1066, CX, Amsterdam, The Netherlands.
authors: Annemieke
authors: A
authors: de Jong
authors: Department of Chemical Immunology, Leiden University Medical Center, Albinusdreef 2, 2333, ZA, Leiden, The Netherlands. h.ovaa@nki.nl.|Division of Cell Biology II, Netherlands Cancer Institute, Plesmanlaan 121, 1066, CX, Amsterdam, The Netherlands. h.ovaa@nki.nl.
authors: Huib
authors: H
authors: Ovaa
mesh_terms: D000345:Affinity Labels; D000818:Animals; D045744:Cell Line, Tumor; D000072017:Deubiquitinating Enzymes / Q000737:chemistry / Q000378:metabolism*; D005456:Fluorescent Dyes; D051379:Mice; D025801:Ubiquitin / Q000737:chemistry*
chemical_list: D000345:Affinity Labels; D005456:Fluorescent Dyes; D025801:Ubiquitin; D000072017:Deubiquitinating Enzymes
keywords: Activity-based protein profiling; Deubiquitinating enzymes; Fluorescent probes; Solid-phase synthesis; Ubiquitin