Abstract
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.
Highlights
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
Mouse cells are resistant to these compounds, including antiproliferative multiple myeloma [18] and thalidomide-associated teratogenicity [19]
Functional conservation among cereblon sequence variants changed to isoleucine in mouse (Ile-391), has been reported to render mouse CRBN unable to degrade Ikaros, Aiolos, and CK1␣ binding through a -hairpin–loop motif that is recognized only when CRBN is complexed with the immunomodulatory drugs [20]
Summary
Comparative analyses of CRBN sequences from representative vertebrate species revealed that Ile-391 is conserved among many nonprimate mammals (mouse, rat, dog, manatee, and opossum), birds (chicken and zebra finch), reptiles (alligator), amphibians (Xenopus), bony fish (zebrafish and spotted gar), and cartilaginous fish (whale shark) (Fig. S1B) [26]. Immunomodulatory compounds bind to a conserved pocket within the C-terminal TBD of CRBN (Fig. S1 and Fig. 2A) [32, 33]. These interactions are governed by hydrogen bonding, aromatic quadrupole, and van der Waals interactions. Analysis of the X-ray crystal structures of CRBN (human (hCRBN), mouse (mCRBN), and chicken (gCRBN)) in complex with thalidomide, Len, and Pom, respectively (Fig. 2A), shows negligible variations of root mean square deviation (RMSD) between the inhibitor poses. Amino acids in mouse CRBN–TBD at Val-380 (equivalent to human Glu-377) and Ile-391 (equivalent to human Val-388) (Fig. 3, A–C) appear to have no relevance in the structure or corresponding immunomodulatory drug– binding interaction based on theoretical modeling. The C366S amino acid mutation was not studied in binding assays, as it is more than 20 Å away from the
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