RRC ID 11043
Author Chou TF, Deshaies RJ.
Title Quantitative cell-based protein degradation assays to identify and classify drugs that target the ubiquitin-proteasome system.
Journal J. Biol. Chem.
Abstract 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.
Volume 286(19)
Pages 16546-54
Published 2011-5-13
DOI 10.1074/jbc.M110.215319
PII M110.215319
PMID 21343295
PMC PMC3089497
MeSH Adenosine Triphosphatases / chemistry Cell Culture Techniques / methods Cell Line Chemistry, Pharmaceutical / methods Green Fluorescent Proteins / chemistry HeLa Cells Humans Inhibitory Concentration 50 Nuclear Proteins / chemistry Proteasome Endopeptidase Complex / chemistry* Proteasome Endopeptidase Complex / metabolism Protein Structure, Tertiary Proteins / chemistry* RNA, Small Interfering / metabolism Technology, Pharmaceutical / methods Ubiquitin / chemistry*
IF 4.011
Times Cited 25
Human and Animal Cells 293T (RCB2202) HeLa (RCB0007)
DNA material pAxCALNLmAREC3 (forward) (RDB02405) GST-Fyn SH3 Fyn SH3 domain (RDB02120) pAxCALNLmSIX2 (reverse) (RDB02402)