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Published May 13, 2011 | Published + Supplemental Material
Journal Article Open

Quantitative Cell-based Protein Degradation Assays to Identify and Classify Drugs That Target the Ubiquitin-Proteasome System


We have generated a set of dual-reporter human cell lines and devised a chase protocol to quantify proteasomal degradation of a ubiquitin fusion degradation (UFD) substrate, a ubiquitin ligase CRL2^(VHL) substrate, and a ubiquitin-independent substrate. Well characterized inhibitors that target different aspects of the ubiquitin-proteasome system can be distinguished by their distinctive patterns of substrate stabilization, enabling assignment of test compounds as inhibitors of the proteasome, ubiquitin chain formation or perception, CRL activity, or the UFD-p97 pathway. We confirmed that degradation of the UFD but not the CRL2^(VHL) or ubiquitin-independent substrates depends on p97 activity. We optimized our suite of assays to establish conditions suitable for high-throughput screening and then validated their performance by screening against 160 cell-permeable protein kinase inhibitors. This screen identified Syk inhibitor III as an irreversible p97/vasolin containing protein inhibitor (IC_(50) = 1.7 μm) that acts through Cys-522 within the D2 ATPase domain. Our work establishes a high-throughput screening-compatible pipeline for identification and classification of small molecules, cDNAs, or siRNAs that target components of the ubiquitin-proteasome system.

Additional Information

© 2011 The American Society for Biochemistry and Molecular Biology, Inc. Free via Creative Commons: CC. Creative Commons Attribution Non-Commercial License applies to Author Choice Articles. Received for publication, December 22, 2010, and in revised form, February 14, 2011 Published, JBC Papers in Press, February 22, 2011. We thank M. Smythe and C. Crews for YU101, A. M. Weissman for PYR-41, Y. Ye for EerI, Millennium Pharmaceuticals for MLN4924, K. Vousden for JNJ26854165, J. Huang for SMER3, and C. C. Wu for 3,4-methylenedioxycinnamic acid (compound 18). We thank P. I. Hanson, A. T. Brunger, and A. L. King for providing plasmids and M. G. Masucci, N. P. Dantuma, R. R. Kopito, and D. Baltimore for providing cell lines. We thank F. Parlati for critical reading of the manuscript; H. Park, R. Oania, and D. Shimoda for technical assistance; and the members of the Molecular Libraries Probe Production Centers Network at The Scripps Research Institute and Kansas University for advice and guidance on the implementation of the ATPase assay and discussions. This work was supported, in whole or in part, by National Institutes of Health Grant R03 MH085687. This work was also supported by the Howard Hughes Medical Institute.

Attached Files

Published - Chou2011p14027J_Biol_Chem.pdf

Supplemental Material - jbc.M110.215319-1.pdf


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