Ubiquitin-specific proximity labeling for the identification of E3 ubiquitin ligase substrates

Protein ubiquitination controls diverse processes within eukaryotic cells, including protein degradation, and is often dysregulated in diseases1. Moreover, protein degraders that redirect ubiquitination activities toward disease targets are an emerging and promising therapeutic class2. Over 600 E3 ubiquitin ligases are expressed in humans3,4, but their substrates remain largely elusive due to a lack of robust methods to identify E3 ligase substrates. Here we report the development of E-STUB (E3 substrate tagging by ubiquitin biotinylation), a ubiquitin-specific proximity labeling method that biotinylates ubiquitinated substrates in proximity to an E3 ligase of interest. E-STUB accurately identifies the direct ubiquitinated targets of protein degraders, including collateral targets and ubiquitylation events that do not exhibit a degradative outcome. It also detects known substrates of E3 ligase cereblon (CRBN) and von Hippel-Lindau (VHL) with high precision. With the ability to elucidate proximal ubiquitination events, E-STUB may facilitate the development of proximity-inducing drugs and act as a generalizable method for E3 substrate mapping.

蛋白质泛素化（Protein ubiquitination）调控真核细胞内的多种生物学过程，包括蛋白质降解，且常在疾病中发生失调¹。此外，将泛素化活性重定向至疾病靶点的蛋白质降解剂（protein degraders）是一类新兴且极具潜力的治疗药物类别²。人类体内表达的E3泛素连接酶（E3 ubiquitin ligases）超过600种³,⁴，但由于缺乏可靠的E3连接酶底物鉴定方法，其底物在很大程度上仍未明确。本研究报道了E-STUB（E3 substrate tagging by ubiquitin biotinylation）的开发，这是一种泛素特异性邻近标记技术，可在目标E3连接酶的邻近范围内对泛素化底物进行生物素标记。E-STUB能够精准鉴定蛋白质降解剂的直接泛素化靶点，包括附带靶点以及不产生降解效应的泛素化事件。该技术还可高精准度地检测E3连接酶脑蛋白（cereblon, CRBN）与希佩尔-林道蛋白（von Hippel-Lindau, VHL）的已知底物。凭借阐明邻近泛素化事件的能力，E-STUB有望推动邻近诱导型药物的开发，并成为一种可通用的E3底物定位方法。