School of Food and Biological Engineering, Engineering Research Center of Bio-process, Ministry of Education, Hefei University of Technology, Hefei, 230009, China.
Tsinghua-Peking Center for Life Sciences, Ministry of Education Key Laboratory of Bioorganic Phosphorus Chemistry and Chemical Biology, Department of Chemistry, Tsinghua University, Beijing, 100084, China.
Angew Chem Int Ed Engl. 2022 Jul 11;61(28):e202203792. doi: 10.1002/anie.202203792. Epub 2022 May 19.
Photocaged, activity-based ubiquitin (Ub) probes (Ub-ABPs) have been developed for the time-resolved probing of deubiquitinating enzyme (DUB) activities, but many Ub-ABPs are still challenging to photocage because their warheads (e.g. propargylamide (PA) or dehydroalanine (Dha)) are difficult to temporally block and activate. Here, we describe a new C-terminal backbone modification strategy for the construction of photocaged Ub-ABPs in which a light-sensitive group is placed at the backbone amide bond of the Ub Gly75. This strategy enabled the facile generation of cell-permeable photocaged Ub-PA and Dha probes that could be activated to capture DUBs after photo-irradiation, and were used to profile DUBs in cells under specially designed conditions (e.g. in cells experiencing oxidative stress) or DUBs with isopeptide linkage selectivity. This backbone modification strategy is anticipated to provide a general solution for the development of photocaged Ub ABPs bearing any warheads for DUB profiling.
光笼型、基于活性的泛素（Ub）探针（Ub-ABPs）已被开发用于检测去泛素化酶（DUB）的时间分辨活性，但许多 Ub-ABPs 仍然难以光笼化，因为它们的弹头（例如炔丙酰胺（PA）或脱氢丙氨酸（Dha））难以暂时阻断和激活。在这里，我们描述了一种新的 C 末端骨架修饰策略，用于构建光笼型 Ub-ABPs，其中在 Ub Gly75 的酰胺键处放置一个光敏感基团。该策略能够方便地生成细胞通透性的光笼型 Ub-PA 和 Dha 探针，这些探针在光照射后可以被激活以捕获 DUBs，并用于在特殊设计的条件下（例如在经历氧化应激的细胞中）或具有异肽键选择性的 DUBs 中进行 DUB 分析。该骨架修饰策略有望为开发带有任何弹头的光笼型 Ub ABPs 用于 DUB 分析提供一种通用的解决方案。
