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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.

SUBMITTER: Pao KC 

PROVIDER: S-EPMC4909137 | biostudies-literature | 2016 May

REPOSITORIES: biostudies-literature

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Probes of ubiquitin E3 ligases enable systematic dissection of parkin activation.

Pao Kuan-Chuan KC   Stanley Mathew M   Han Cong C   Lai Yu-Chiang YC   Murphy Paul P   Balk Kristin K   Wood Nicola T NT   Corti Olga O   Corvol Jean-Christophe JC   Muqit Miratul M K MM   Virdee Satpal S  

Nature chemical biology 20160307 5


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 phosphor  ...[more]

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