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

泛素(Ub)对细胞蛋白质的翻译后修饰参与了细胞生理的多个方面，如通过蛋白酶体降解蛋白质、DNA修复和膜运输。脱辅酶从附着在底物蛋白上的多Ub链上释放出一个Ub部分。在目前的研究中，我们研究了脱泛素化酶的酶学特性的新的研究工具。Ub-颗粒酶B(Ub-GRb)是一种N-末端的融合成熟颗粒酶B，在杆状病毒系统中获得表达。通过使用Ub-GRB，我们证明了人泛素特异性蛋白水解酶47具有活性。此外，我们还探索了氧化应激敏感的脱泛素化酶，发现泛素C末端水解酶-L3利用基于Ub活性的探针Ub-乙烯基砜对活性氧物种敏感。

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.