Shu Xin, Liao Qing-Qing, Li Shang-Tong, Liu Lu, Zhang Xiajun, Zhou Lianqi, Zhang Long, Coin Irene, Wang Lei, Wu Haifan, Yang Bing
Zhejiang Provincial Key Laboratory for Cancer Molecular Cell Biology, Life Sciences Institute, Zhejiang University, Hangzhou, Zhejiang 310058, China.
Cancer Center, Zhejiang University, Hangzhou, Zhejiang 310058, China.
Anal Chem. 2023 Jan 17;95(2):846-853. doi: 10.1021/acs.analchem.2c03270. Epub 2023 Jan 3.
Post-translational modification of proteins by Ubiquitin (Ub) and Ubiquitin-like proteins (Ubls) can be reversed by deconjugating enzymes, which have been implicated in different pathways and associated with various human diseases. To understand the activity and dynamics of deconjugating enzymes, multiple synthetic and semi-synthetic Ub/Ubl probes have been developed, and some of them have been applied to screen inhibitors of deconjugating enzymes. Since these Ub/Ubl probes are generally not cell-permeable, different strategies have been developed to deliver Ub/Ubl probes to live cells. However, till now, no Ub/Ubl probes can be expressed in live cells to directly report on the activities of deconjugating enzymes in the most relevant cellular environment. Here, we genetically encoded cross-linkable Ub/Ubl probes in live and HEK293T cells. These probes can cross-link with deconjugating enzymes and . Using these Ub probes combined with mass spectrometry, we have successfully identified endogenous deconjugating enzymes in live cells. We believe that these genetically encoded Ub/Ubl probes are valuable for investigating biological functions of deconjugating enzymes in physiological environments.
泛素（Ub）和类泛素蛋白（Ubls）对蛋白质的翻译后修饰可被去共轭酶逆转，这些酶涉及不同途径并与多种人类疾病相关。为了解去共轭酶的活性和动力学，已开发出多种合成和半合成的Ub/Ubl探针，其中一些已用于筛选去共轭酶的抑制剂。由于这些Ub/Ubl探针通常不能穿透细胞，因此已开发出不同策略将Ub/Ubl探针递送至活细胞。然而，迄今为止，尚无Ub/Ubl探针可在活细胞中表达以直接报告在最相关细胞环境中去共轭酶的活性。在此，我们在活细胞和HEK293T细胞中对可交联的Ub/Ubl探针进行了基因编码。这些探针可与去共轭酶和交联。使用这些Ub探针结合质谱分析，我们已成功鉴定出活细胞中的内源性去共轭酶。我们认为这些基因编码的Ub/Ubl探针对于研究去共轭酶在生理环境中的生物学功能具有重要价值。
