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A simple cell based assay to measure Parkin activity
Author(s) -
Morrison Ewan,
Thompson John,
Williamson Sally J. M.,
Cheetham Michael E.,
Robinson Philip A.
Publication year - 2011
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.2010.07113.x
Subject(s) - parkin , mitophagy , ubiquitin ligase , ubiquitin , biology , proteostasis , microbiology and biotechnology , programmed cell death , proteasome , lactacystin , mitochondrion , proteasome inhibitor , biochemistry , autophagy , apoptosis , parkinson's disease , medicine , disease , pathology , gene
J. Neurochem. (2011) 116 , 342–349. 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.