Biochemical and physiological studies on deubiquitinating enzymes based on an analysis of the enzymatic activity dynamics

泛素（ubiquitin，Ub）对细胞蛋白质的翻译后修饰涉及细胞生理学的各个方面，如蛋白酶体降解、DNA修复和膜运输等。去泛素化酶从附着在底物蛋白上的polyUb链释放Ub部分。在目前的研究中，我们研究了新的研究工具，酶的特性去泛素化酶。Ub-颗粒酶B（Ub-GrB）是一种N-末端融合的成熟颗粒酶B。通过使用Ub-GrB，我们表明人泛素特异性蛋白酶47是有活性的。此外，我们探讨了氧化应激敏感的去泛素化酶，并发现泛素C-末端水解酶-L3是敏感的活性氧物种，利用Ub活性为基础的探针，Ub-乙烯砜。

Post-translational modification of cellular proteins by ubiquitin (Ub) is involved in various aspects of cell physiology, such as protein degradation via proteasome, DNA repair, and membrane trafficking. Deubiqutinating enzymes liberate a Ub moiety from polyUb chains attached on substrate proteins. In the current study, we investigated novel research tools for the enzymatic characterization of deubiquitinating enzymes. Ub-granzyme B (Ub-GrB), an N-terminal Ub fusion mature granzyme B was expressed in a baculovirus system. By employing Ub-GrB, we showed that human ubiquitin specific protease 47 is active. In addition, we explored oxidative stress-sensitive deubiquitinating enzymes, and found that ubiquitin C-terminal hydrolase-L3 is susceptible to reactive oxygen species by utilizing a Ub activity-based probe, Ub-vinyl sulfone.