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A novel luminescence-based high-throughput approach for cellular resolution of protein ubiquitination using tandem ubiquitin binding entities (TUBEs).

Akinjiyan, Favour and Fazal, Aleem and Hild, Marc and Beckwith, Rohan and Paulk, Josh and Carbonneau, Seth (2020) A novel luminescence-based high-throughput approach for cellular resolution of protein ubiquitination using tandem ubiquitin binding entities (TUBEs). SLAS DISCOVERY: Advancing the Science of Drug Discovery, 25 (4). pp. 350-360. ISSN 2472-55522472-5560

Abstract

Protein turnover is highly regulated by the post-translational process of ubiquitination. Deregulation of the ubiquitin proteasome system (UPS) has been implicated in cancer and neurodegenerative diseases, and modulating this system has proven to be a viable approach for therapeutic intervention. Development of novel technologies that enable high-throughput studies of substrate protein ubiquitination is essential for drug discovery in the UPS. Conventional approaches for studying ubiquitination either require high amounts of starting protein or rely on exogenous or modified ubiquitin moieties and thus limiting their utility. In order to circumvent these issues, we developed a high-throughput live-cell assay which combines the NanoBiT luminescence-based technology with tandem ubiquitin-binding entities (TUBE) to resolve substrate ubiquitination. To demonstrate the effectiveness and utility of this assay, we studied the compound-induced ubiquitination of G To S Phase Transition 1 (GSPT1) protein. Using this new assay, we characterized compounds with varying levels of GSPT1 ubiquitination activity. This method provides a novel cell based approach for assaying substrate ubiquitination in living cells that can be adapted to study the kinetics of ubiquitin transfer onto a substrate protein of interest. In addition, our results show that this approach is portable for studying ubiquitination of target proteins with varying functions.

Item Type: Article
Keywords: ubiquitin, G To S Phase Transition 1 Protein (GSPT1), luminescence, TUBE domain, cellular assay
Date Deposited: 09 Apr 2020 00:45
Last Modified: 09 Apr 2020 00:45
URI: https://oak.novartis.com/id/eprint/36052

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