Ligand-mediated protein degradation reveals functional conservation among sequence variants of the CUL4-type E3 ligase substrate receptor cereblon

• Published in: The Journal of biological chemistry Vol. 293; no. 16; pp. 6187 - 6200
• Main Authors: Akuffo, Afua A., Alontaga, Aileen Y., Metcalf, Rainer, Beatty, Matthew S., Becker, Andreas, McDaniel, Jessica M., Hesterberg, Rebecca S., Goodheart, William E., Gunawan, Steven, Ayaz, Muhammad, Yang, Yan, Karim, Md Rezaul, Orobello, Morgan E., Daniel, Kenyon, Guida, Wayne, Yoder, Jeffrey A., Rajadhyaksha, Anjali M., Schönbrunn, Ernst, Lawrence, Harshani R., Lawrence, Nicholas J., Epling-Burnette, Pearlie K.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 20-04-2018; American Society for Biochemistry and Molecular Biology
• Subjects: Adaptor Proteins, Signal Transducing; Animals; Azepines - pharmacology; bromodomain-containing protein 4 (BRD4); Cell Cycle Proteins; Cell Line, Tumor; Conserved Sequence; Cullin Proteins - metabolism; E3 ubiquitin ligase; Humans; Immunologic Factors - metabolism; Immunologic Factors - pharmacology; immunology; Lenalidomide - pharmacology; Ligands; Mice; Molecular Probes; mouse; multiple myeloma; Nuclear Proteins - drug effects; Nuclear Proteins - metabolism; Peptide Hydrolases - chemistry; Peptide Hydrolases - metabolism; proteasome; Proteasome Endopeptidase Complex - metabolism; Protein Structure and Folding; Proteolysis; T-Lymphocytes - metabolism; Thalidomide - analogs & derivatives; Thalidomide - metabolism; Thalidomide - pharmacology; Transcription Factors - drug effects; Transcription Factors - metabolism; Triazoles - pharmacology; ubiquitin; Ubiquitin - metabolism; Ubiquitin-Protein Ligases - metabolism; bromodomain-containing protein 4 (BRD4); mouse; proteasome; multiple myeloma; E3 ubiquitin ligase; immunology; ubiquitin
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M117.816868

Upon binding to thalidomide and other immunomodulatory drugs, the E3 ligase substrate receptor cereblon (CRBN) promotes proteosomal destruction by engaging the DDB1–CUL4A–Roc1–RBX1 E3 ubiquitin ligase in human cells but not in mouse cells, suggesting that sequence variations in CRBN may cause its inactivation. Therapeutically, CRBN engagers have the potential for broad applications in cancer and immune therapy by specifically reducing protein expression through targeted ubiquitin-mediated degradation. To examine the effects of defined sequence changes on CRBN’s activity, we performed a comprehensive study using complementary theoretical, biophysical, and biological assays aimed at understanding CRBN’s nonprimate sequence variations. With a series of recombinant thalidomide-binding domain (TBD) proteins, we show that CRBN sequence variants retain their drug-binding properties to both classical immunomodulatory drugs and dBET1, a chemical compound and targeting ligand designed to degrade bromodomain-containing 4 (BRD4) via a CRBN-dependent mechanism. We further show that dBET1 stimulates CRBN’s E3 ubiquitin–conjugating function and degrades BRD4 in both mouse and human cells. This insight paves the way for studies of CRBN-dependent proteasome-targeting molecules in nonprimate models and provides a new understanding of CRBN’s substrate-recruiting function.