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.
Subject(s)
Adaptor Proteins, Signal Transducing/genetics , Biological Assay , DNA-Binding Proteins/genetics , Hepatocytes/metabolism , Lymphocytes/metabolism , Transcription Factors/genetics , Ubiquitin-Protein Ligases/genetics , Adaptor Proteins, Signal Transducing/metabolism , Biotinylation , Cell Line, Tumor , DNA-Binding Proteins/metabolism , Gene Expression Regulation , HEK293 Cells , Hepatocytes/cytology , Hepatocytes/drug effects , Humans , Immunologic Factors/pharmacology , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Lymphocytes/cytology , Lymphocytes/drug effects , Neurons/cytology , Neurons/drug effects , Neurons/metabolism , Protein Binding , Protein Interaction Mapping , Proteolysis/drug effects , Receptor, Fibroblast Growth Factor, Type 1/genetics , Receptor, Fibroblast Growth Factor, Type 1/metabolism , Substrate Specificity , Sulfonamides/pharmacology , Transcription Factors/metabolism , Ubiquitin-Protein Ligases/metabolismABSTRACT
Regulating the amount of proteins in living cells is a powerful approach for understanding the functions of the proteins. Immunomodulatory drugs (IMiDs) induce the degradation of neosubstrates by interacting with celebron (CRBN) in the cullin E3 ubiquitin ligase complex (CRL4CRBN). Here, we developed the IMiD-dependent Sal-like protein 4 (SALL4) degron (S4D) system for chemical protein knockdown. In transient assays, an N- or C-terminal S4D tag induced the degradation of proteins localized to various subcellular compartments, including the plasma membrane. The activity of luciferase-S4D was reduced by 90% within 3 h of IMiD treatment. IMiD treatment reduced the expression of endogenous S4D-fused RelA and IκBα in knock-in (KI) experiments. Interestingly, the IκBα knockdown suggested that there may be another, unknown mechanism for RelA translocation to the nucleus. Furthermore, 5-hydroxythalidomide as a thalidomide metabolite specifically degradated S4D-tagged protein. These results indicate that the S4D system is a useful tool for cellular biology.