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

泛素（Ub）对细胞蛋白的翻译后修饰涉及细胞生理学的各个方面，如蛋白酶体的蛋白质降解、DNA修复和膜运输。去泛素酶从附着在底物蛋白上的聚脲链上释放一个Ub片段。在目前的研究中，我们研究了新的研究工具来酶表征去泛素化酶。Ub-granzyme B （Ub- grb）是一种n端Ub融合成熟的颗粒酶B，在杆状病毒系统中表达。通过使用Ub-GrB，我们发现人泛素特异性蛋白酶47具有活性。此外，我们探索了氧化应激敏感的去泛素化酶，并发现泛素c端水解酶- l3对活性氧敏感，利用基于Ub活性的探针，Ub乙烯基砜。

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.