A proximity biotinylation-based approach to identify protein-E3 ligase interactions induced by PROTACs and molecular glues

Abstract

Proteolysis-targeting chimaeras (PROTACs) as well as molecular glues such as immunomodulatory drugs (IMiDs) and indisulam are drugs that induce interactions between substrate proteins and an E3 ubiquitin ligases for targeted protein degradation. Here, we develop a workflow based on proximity-dependent biotinylation by AirID to identify drug-induced neo-substrates of the E3 ligase cereblon (CRBN). Using AirID-CRBN, we detect IMiD-dependent biotinylation of CRBN neo-substrates in vitro and identify biotinylated peptides of well-known neo-substrates by mass spectrometry with high specificity and selectivity. Additional analyses reveal ZMYM2 and ZMYM2-FGFR1 fusion protein—responsible for the 8p11 syndrome involved in acute myeloid leukaemia—as CRBN neo-substrates. Furthermore, AirID-DCAF15 and AirID-CRBN biotinylate neo-substrates targeted by indisulam and PROTACs, respectively, suggesting that this approach has the potential to serve as a general strategy for characterizing drug-inducible protein–protein interactions in cells.

Discovery of CRBN as a target of thalidomide: a breakthrough for progress in the development of protein degraders

“…ZMYM2–FGFR1 is a pomalidomide- and avadomide-dependent neosubstrate of CRL4 CRBN . 12,13 The binding mode of ZMYM2 to CRBN is similar to that of Ikaros and Aiolos, but its drug specificity is different, with ZMYM2 showing strong preference to avadomide. Concordantly, avadomide exhibits antiproliferative activity in vitro in bone marrow samples derived from patients with hematological malignancies harboring the t(8;13)(p11;q12) translocation.…”

“…1 CRBN is a substrate recognition receptor for Cullin 4 RING E3 ubiquitin ligase (CRL4); the binding of thalidomide derivatives to CRBN triggers the recruitment of non-native substrates to CRL4 CRBN and their subsequent proteasomal degradation. [2][3][4][5][6][7][8][9][10][11][12][13] Thalidomide derivatives are the first, and currently the only, protein degraders with demonstrated clinical benefit.…”

Methods to characterize and discover molecular degraders in cells

“…126 Not surprisingly, they biotinylated IKZF1 and SALL4 as known neosubstrates for pomalidomide along with other parts of the CRL4 complex, including CUL4 and RBX1. In their more recent studies, 127 using AirID–CRBN with different degraders and cell lines, they discovered novel protein targets, including ZMYM2.…”

Optimized Workflow for Enrichment and Identification of Biotinylated Peptides Using Tamavidin 2-REV for BioID and Cell Surface Proteomics

“…Tamavidin 2-REV is an engineered version of Tamavidin 2 that acquired reversible biotin-binding capability by mutating Ser36 that forms a hydrogen bond with biotin. , Because biotinylated peptides can be mildly and specifically eluted from Tamavidin 2-REV beads by adding excess free biotin, the enrichment efficiency is higher than that of anti-biotin antibody beads. However, the low level of protein biotinylation in BioID-expressing cells meant that large amounts of cellular proteins were required to enable identification of biotinylated peptides by mass spectrometry. , Furthermore, many contaminant ions derived from our enrichment process were observed, which may lead to damage to the mass spectrometer. Therefore, it is important to optimize the enrichment method for biotinylated peptides, analogous to the various enrichment methods that have been extensively optimized for phosphorylated peptides.…”