title: Probes of ubiquitin E3 ligases enable systematic dissection of parkin activation.
abstract: E3 ligases represent an important class of enzymes, yet there are currently no chemical probes for profiling their activity. We develop a new class of activity-based probe by re-engineering a ubiquitin-charged E2 conjugating enzyme and demonstrate the utility of these probes by profiling the transthiolation activity of the RING-in-between-RING (RBR) E3 ligase parkin in vitro and in cellular extracts. Our study provides valuable insight into the roles, and cellular hierarchy, of distinct phosphorylation events in parkin activation. We also profile parkin mutations associated with patients with Parkinson's disease and demonstrate that they mediate their effect largely by altering transthiolation activity. Furthermore, our probes enable direct and quantitative measurement of endogenous parkin activity, revealing that endogenous parkin is activated in neuronal cell lines (≥75%) in response to mitochondrial depolarization. This new technology also holds promise as a novel biomarker of PINK1-parkin signaling, as demonstrated by its compatibility with samples derived from individuals with Parkinson's disease.
authors.affiliation: Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, Dundee, UK.
authors.forename: Kuan-Chuan
authors.initials: KC
authors.lastname: Pao
authors.affiliation: Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, Dundee, UK.
authors.forename: Mathew
authors.identifier: http://orcid.org/0000-0002-0631-8306
authors.initials: M
authors.lastname: Stanley
authors.affiliation: Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, Dundee, UK.
authors.forename: Cong
authors.initials: C
authors.lastname: Han
authors.affiliation: Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, Dundee, UK.
authors.forename: Yu-Chiang
authors.initials: YC
authors.lastname: Lai
authors.affiliation: Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, Dundee, UK.
authors.forename: Paul
authors.initials: P
authors.lastname: Murphy
authors.affiliation: Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, Dundee, UK.
authors.forename: Kristin
authors.initials: K
authors.lastname: Balk
authors.affiliation: Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, Dundee, UK.
authors.forename: Nicola T
authors.initials: NT
authors.lastname: Wood
authors.affiliation: Sorbonne Universités, Université Pierre et Marie Curie; Institut National de la Santé et de la Recherche Médicale, Unité 1127, Centre d'investigation Clinique 1422; Centre National de la Recherche Scientifique, Unité 7225; Assistance Publique Hôpitaux de Paris; Institut du Cerveau et de la Moelle, Paris, France.
authors.forename: Olga
authors.initials: O
authors.lastname: Corti
authors.affiliation: Sorbonne Universités, Université Pierre et Marie Curie; Institut National de la Santé et de la Recherche Médicale, Unité 1127, Centre d'investigation Clinique 1422; Centre National de la Recherche Scientifique, Unité 7225; Assistance Publique Hôpitaux de Paris; Institut du Cerveau et de la Moelle, Paris, France.
authors.forename: Jean-Christophe
authors.initials: JC
authors.lastname: Corvol
authors.affiliation: Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, Dundee, UK.|School of Medicine, University of Dundee, Dundee, UK.
authors.forename: Miratul M K
authors.initials: MM
authors.lastname: Muqit
authors.affiliation: Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, Dundee, UK.
authors.forename: Satpal
authors.identifier: http://orcid.org/0000-0002-7052-9802
authors.initials: S
authors.lastname: Virdee
mesh_terms: D005347:Fibroblasts / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D009154:Mutation; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*
chemical_list: D044767:Ubiquitin-Protein Ligases; C111567:parkin protein