Damianou Andreas, Jones Hannah B L, Grigoriou Athina, Akbor Mohammed A, Jenkins Edward, Charles Philip D, Vendrell Iolanda, Davis Simon, Kessler Benedikt M
Target Discovery Institute, Centre for Medicines Discovery, Nuffield Department of Medicine, University of Oxford, Roosevelt Drive, OX3 7FZ Oxford, UK; Chinese Academy for Medical Sciences Oxford Institute, Nuffield Department of Medicine, University of Oxford, Roosevelt Drive, OX3 7FZ Oxford, UK.
Target Discovery Institute, Centre for Medicines Discovery, Nuffield Department of Medicine, University of Oxford, Roosevelt Drive, OX3 7FZ Oxford, UK.
Cell Chem Biol. 2025 May 15;32(5):736-751.e8. doi: 10.1016/j.chembiol.2025.04.004. Epub 2025 May 5.
The growing interest in deubiquitinases (DUBs) as drug targets for modulating critical molecular pathways in disease is fueled by the discovery of their specific cellular roles. A crucial aspect of this fact is the identification of DUB substrates. While mass spectrometry-based proteomic methods can be used to study global changes in cellular ubiquitination following DUB activity perturbation, these datasets often include indirect and downstream ubiquitination events. To enrich for the direct substrates of DUB enzymes, we have developed a proximal-ubiquitome workflow that combines proximity labeling methodology (ascorbate peroxidase-2 [APEX2]) with subsequent ubiquitination enrichment based on the K-ε-GG motif. We applied this technology to identify altered ubiquitination events in the vicinity of the DUB ubiquitin-specific protease 30 (USP30) upon its inhibition. Our findings reveal ubiquitination events previously associated with USP30 on TOMM20 and FKBP8, as well as the candidate substrate LETM1, which is deubiquitinated in a USP30-dependent manner.
去泛素化酶（DUBs）作为调节疾病关键分子途径的药物靶点，其细胞特异性作用的发现激发了人们对它日益增长的兴趣。这一事实的一个关键方面是DUB底物的鉴定。虽然基于质谱的蛋白质组学方法可用于研究DUB活性扰动后细胞泛素化的整体变化，但这些数据集通常包括间接和下游泛素化事件。为了富集DUB酶的直接底物，我们开发了一种近端泛素组工作流程，该流程将邻近标记方法（抗坏血酸过氧化物酶-2 [APEX2]）与基于K-ε-GG基序的后续泛素化富集相结合。我们应用这项技术来鉴定DUB泛素特异性蛋白酶30（USP30）被抑制后其附近改变的泛素化事件。我们的研究结果揭示了先前与USP30相关的泛素化事件，这些事件发生在TOMM20和FKBP8上，以及候选底物LETM1，它以USP30依赖的方式去泛素化。
