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7144481 
Journal Article 
Novel approach for characterizing ubiquitin E3 ligase function 
Marblestone, JG; Suresh Kumar, KG; Eddins, MJ; Leach, CA; Sterner, DE; Mattern, MR; Nicholson, B; , 
2010 
Yes 
Journal of Biomolecular Screening
ISSN: 1087-0571
EISSN: 1552-454X 
SAGE PUBLICATIONS INC 
THOUSAND OAKS 
1220-1228 
English 
20864734 
WOS:000284836900005 
The ubiquitin-proteasome system is central to the regulation of numerous cellular events, and dysregulation may lead to disease pathogenesis. E3 ubiquitin ligases typically function in concert with E1 and E2 enzymes to recruit specific substrates, thereby coordinating their ubiquitylation and subsequent proteasomal degradation or cellular activity. E3 ligases have been implicated in a wide range of pathologies, and monitoring their activity in a rapid and cost-effective manner would be advantageous in drug discovery. The relative lack of high-throughput screening (HTS)-compliant E3 ligase assays has significantly hindered the discovery of E3 inhibitors. Herein, the authors describe a novel HTS-compliant E3 ligase assay platform that takes advantage of a ubiquitin binding domain's inherent affinity for polyubiquitin chains, permitting the analysis of ubiquitin chain formation in an E3 ligase-dependent manner. This assay has been used successfully with members of both the RING and HECT families, demonstrating the platform's broad utility for analyzing a wide range of E3 ligases. The utility of the assay platform is demonstrated by the identification of inhibitors of the E3 ligase CARP2. As the number of E3 ligases associated with various disease states increases, the ability to quantitate the activity of these enzymes in an expeditious manner becomes imperative in drug discovery.