title: A cascading activity-based probe sequentially targets E1-E2-E3 ubiquitin enzymes.
abstract: Post-translational modifications of proteins with ubiquitin (Ub) and ubiquitin-like modifiers (Ubls), orchestrated by a cascade of specialized E1, E2 and E3 enzymes, control a wide range of cellular processes. To monitor catalysis along these complex reaction pathways, we developed a cascading activity-based probe, UbDha. Similarly to the native Ub, upon ATP-dependent activation by the E1, UbDha can travel downstream to the E2 (and subsequently E3) enzymes through sequential trans-thioesterifications. Unlike the native Ub, at each step along the cascade, UbDha has the option to react irreversibly with active site cysteine residues of target enzymes, thus enabling their detection. We show that our cascading probe 'hops' and 'traps' catalytically active Ub-modifying enzymes (but not their substrates) by a mechanism diversifiable to Ubls. Our founder methodology, amenable to structural studies, proteome-wide profiling and monitoring of enzymatic activity in living cells, presents novel and versatile tools to interrogate Ub and Ubl cascades.
authors.affiliation: Division of Cell Biology, Netherlands Cancer Institute, Amsterdam, the Netherlands.
authors.forename: Monique P C
authors.initials: MP
authors.lastname: Mulder
authors.affiliation: Division of Cell Biology, Netherlands Cancer Institute, Amsterdam, the Netherlands.
authors.forename: Katharina
authors.initials: K
authors.lastname: Witting
authors.affiliation: Division of Cell Biology, Netherlands Cancer Institute, Amsterdam, the Netherlands.
authors.forename: Ilana
authors.initials: I
authors.lastname: Berlin
authors.affiliation: Division of Protein and Nucleic Acid Chemistry, Medical Research Council Laboratory of Molecular Biology, Cambridge, UK.
authors.forename: Jonathan N
authors.initials: JN
authors.lastname: Pruneda
authors.affiliation: Department of Structural Biology, St. Jude Children's Research Hospital, Memphis, Tennessee, USA.
authors.forename: Kuen-Phon
authors.identifier: 0000-0002-1525-6004
authors.initials: KP
authors.lastname: Wu
authors.affiliation: Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, the Netherlands.
authors.forename: Jer-Gung
authors.initials: JG
authors.lastname: Chang
authors.affiliation: Division of Cell Biology, Netherlands Cancer Institute, Amsterdam, the Netherlands.
authors.forename: Remco
authors.initials: R
authors.lastname: Merkx
authors.affiliation: Division of Immunology, Department of Biology, University of Konstanz, Konstanz, Germany.
authors.forename: Johanna
authors.initials: J
authors.lastname: Bialas
authors.affiliation: Division of Immunology, Department of Biology, University of Konstanz, Konstanz, Germany.
authors.forename: Marcus
authors.initials: M
authors.lastname: Groettrup
authors.affiliation: Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, the Netherlands.
authors.forename: Alfred C O
authors.initials: AC
authors.lastname: Vertegaal
authors.affiliation: Department of Structural Biology, St. Jude Children's Research Hospital, Memphis, Tennessee, USA.|Howard Hughes Medical Institute, Memphis, Tennessee, USA.
authors.forename: Brenda A
authors.initials: BA
authors.lastname: Schulman
authors.affiliation: Division of Protein and Nucleic Acid Chemistry, Medical Research Council Laboratory of Molecular Biology, Cambridge, UK.
authors.forename: David
authors.initials: D
authors.lastname: Komander
authors.affiliation: Division of Cell Biology, Netherlands Cancer Institute, Amsterdam, the Netherlands.
authors.forename: Jacques
authors.initials: J
authors.lastname: Neefjes
authors.affiliation: Division of Cell Biology, Netherlands Cancer Institute, Amsterdam, the Netherlands.
authors.forename: Farid
authors.initials: F
authors.lastname: El Oualid
authors.affiliation: Division of Cell Biology, Netherlands Cancer Institute, Amsterdam, the Netherlands.
authors.forename: Huib
authors.initials: H
authors.lastname: Ovaa
mesh_terms: D006367:HeLa Cells; D006801:Humans; D008958:Models, Molecular; D015335:Molecular Probes / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D015394:Molecular Structure; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D025801:Ubiquitin / Q000378:metabolism; D044764:Ubiquitin-Activating Enzymes / Q000037:antagonists & inhibitors* / Q000378:metabolism
chemical_list: D015335:Molecular Probes; D025801:Ubiquitin; D044764:Ubiquitin-Activating Enzymes