Abstract 3885: A ubiquitin-specific proximity labeling method to study drug-induced ubiquitylation

Abstract Targeted protein degradation is a promising therapeutic paradigm that offers new ways to disrupt protein functions. Currently, most studies that characterize the pharmacological effects of protein degraders utilize target-specific assays and global proteomics. However, information on the direct molecular consequence of protein degraders, i.e., induced protein ubiquitylation, is often lacking. Here we report the development of E-STUB (E3-substrate tagging by ubiquitin biotinylation), a ubiquitin-specific proximity labeling method that identifies ubiquitylated substrates in proximity to an E3 ligase of interest. We demonstrated that E-STUB can accurately identify the ubiquitylated targets induced by protein degraders in both CRBN and VHL systems—two of the most utilized E3 ligases by heterobifunctional degraders—and is likely generalizable to other E3 ligases. While we observed a high level of consistency between ubiquitylation and degradation for the induced substrates, E-STUB also detected ubiquitylation of non-degraded targets, providing novel and complementary insights to global proteomics. We also envision that E-STUB could be specifically used for the development of chemical inducers of proximity that catalyze non-degradative ubiquitylation events. In summary, we expect that the E-STUB approach can be widely useful for studying the principles of chemically induced ubiquitylation and the advancement of targeted protein degradation. Citation Format: Hai-Tsang Huang, Ryan J. Lumpkin, Ryan W. Tsai, Katherine A. Donovan, Shuyao Su, Xu Zhao, James Chen, Eric S. Fischer, William R. Sellers. A ubiquitin-specific proximity labeling method to study drug-induced ubiquitylation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3885.