Millipore Technical Publications |
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Development of Novel UbiquitinProfiler™ Activity Assays for the E3 Ligase Parkin | ||
| Lit No: | PS3377EN00 | |
| Year: | 2012 | |
| Introduction Dysregulation of ubiquitination pathways is linked with a number of human diseases, and components of these pathways have emerged as a promising class of targets for drug discovery. However, the development of new therapeutics has been hindered by the complexity of the systems involved and a lack of suitable screening technologies. In particular, robust, scalable, and biologically-relevant assays are needed to support inhibitor screening and counter-screening campaigns to target ubiquitination pathways. Parkin is an E3 ubiquitin ligase belonging to the RBR (RING – In Between RING – RING) family. Mutations in Parkin that result in a loss-of-function are the most frequent cause of familial Parkinson’s disease and are responsible for a large percentage of autosomal recessive juvenile parkinsonism (AR-JP). In addition to its auto-ubiquitination, Parkin can also ubiquitinate various substrates, including the CDK2-interacting protein cyclin E1, the α-synuclein-interacting protein synphilin-1, the synaptic vesicle-associated CDC-rel1 and the p38/JTV-1 subunit of aminoacyl-tRNA synthetase. Ubiquitination of substrates occurs in conjunction with the E2 enzymes UbcH7, UbcH8 or UbcH13/Uev1. Here, we describe the development of robust, cost-effective, and biologically-relevant assays to investigate the ubiquitination activity of Parkin towards itself and the p38/JTV-1 substrate. These assays rely on proprietary EMD Millipore enzymes, utilize a sensitive electrochemiluminescence (ECL) detection platform, and are validated for high-throughput small molecule testing. | |
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