title: A simple cell based assay to measure Parkin activity.
abstract: Parkin is an ubiquitin-protein ligase mutated in Autosomal Recessive - Juvenile Parkinsonism. Here, we describe a cell-based assay to measure Parkin's ubiquitin-protein ligase activity. It relies on the ability of Parkin to recognise depolarised mitochondria and exploits a cell line where Parkin expression is inducible. In these cells, Parkin expression promotes mitophagy and accelerates cell death in response to mitochondrial depolarisers. Time-lapse imaging confirmed cell death and revealed increased perinuclear mitochondrial clustering following induction of Parkin expression in cells exposed to carbonyl cyanide m-chlorophenylhydrazone. Similar effects were not observed with α-synuclein or DJ-1, other proteins associated with the development of Parkinson's disease, confirming the specificity of the assay. We have used this assay to demonstrate that ligase-defective Parkin mutants are inactive, and cellular proteasomal activity (using the proteasomal inhibitors MG132, clasto-lactacystin β-lactone and epoxomicin) is essential for the Parkin mediated effect. As the assay is suitable for high-throughput screening, it has the potential to identify novel proteostasis compounds that stimulate the activity of Parkin mutants for therapeutic purposes, to identify modulators of kinase activities that impact on Parkin function, and to act as a functional read-out in reverse genetics screens aimed at identifying modifiers of Parkin function during mitophagy.
authors.affiliation: Section of Ophthalmology and Neuroscience, Leeds Institute for Molecular Medicine, University of Leeds, Leeds, UK.
authors.forename: Ewan
authors.initials: E
authors.lastname: Morrison
authors.forename: John
authors.initials: J
authors.lastname: Thompson
authors.forename: Sally J M
authors.initials: SJ
authors.lastname: Williamson
authors.forename: Michael E
authors.initials: ME
authors.lastname: Cheetham
authors.forename: Philip A
authors.initials: PA
authors.lastname: Robinson
mesh_terms: D015153:Blotting, Western / Q000379:methods; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D016923:Cell Death / Q000187:drug effects / Q000502:physiology; D057809:HEK293 Cells; D006801:Humans; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000187:drug effects / Q000378:metabolism; D059008:Time-Lapse Imaging / Q000379:methods; D044767:Ubiquitin-Protein Ligases / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D014475:Uncoupling Agents / Q000494:pharmacology; D051844:alpha-Synuclein / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D014475:Uncoupling Agents; D051844:alpha-Synuclein; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D044767:Ubiquitin-Protein Ligases; C111567:parkin protein; D046988:Proteasome Endopeptidase Complex