Protein ubiquitylation controls diverse processes within eukaryotic cells, including protein degradation, and is often dysregulated in disease. Moreover, small-molecule degraders that redirect ubiquitylation activities toward disease targets are an emerging and promising therapeutic class. Over 600 E3 ubiquitin ligases are expressed in humans, but their substrates remain largely elusive, necessitating the development of new methods for their discovery. Here we report the development of E3-substrate tagging by ubiquitin biotinylation (E-STUB), a ubiquitin-specific proximity labeling method that biotinylates ubiquitylated substrates in proximity to an E3 ligase of interest. E-STUB accurately identifies the direct ubiquitylated targets of protein degraders, including collateral targets and ubiquitylation events that do not lead to substrate degradation. It also detects known substrates of E3 ligase CRBN and VHL with high specificity. With the ability to elucidate proximal ubiquitylation events, E-STUB may facilitate the development of proximity-inducing therapeutics and act as a generalizable method for E3-substrate mapping.

蛋白质泛素化控制着真核细胞内的多种过程，包括蛋白质降解，并且在疾病中常常失调。此外，将泛素化活性转向疾病靶点的小分子降解剂是一种新兴且有前途的治疗类别。超过 600 种 E3 泛素连接酶在人类中表达，但它们的底物在很大程度上仍然难以捉摸，因此需要开发新的方法来发现它们。在此，我们报告了通过泛素生物素化 (E-STUB) 进行 E3 底物标记的进展，这是一种泛素特异性邻近标记方法，可对感兴趣的 E3 连接酶附近的泛素化底物进行生物素化。 E-STUB 准确识别蛋白质降解剂的直接泛素化靶标，包括附带靶标和不会导致底物降解的泛素化事件。它还以高特异性检测 E3 连接酶 CRBN 和 VHL 的已知底物。凭借阐明近端泛素化事件的能力，E-STUB 可以促进邻近诱导疗法的开发，并作为 E3 底物作图的通用方法。