Abstract: Ubiquitin C-terminal hydropase-L3 (UCHL3), an important member of the ubiquitin C-terminal hydrolases (UCHs) family, is involved in DNA damage repair and other processes. Previous studies showed that UCHL3 can cleave C-terminal conjugate of ubiquitin with 7-amino-4-methylcoumarin (Ub-AMC) in vitro and the phosphorylation of Ser75 obviously promotes the cleavage activity of UCHL3 towards poly-ubiquitin chains in vivo. But this regulation has not been corroborated by any biochemical data in vitro so far. Based on this situation, wild-type UCHL3 (UCHL3WT) and simulated phosphorylated UCHL3 protein (UCHL3S75E) were prepared by QuikChange site-directed mutagenesis technique and step-wise chromatography methods. The effect of phosphorylation at Ser75 on the poly-ubiquitin chains cleavage activity of UCHL3 in vitro was studied. The results showed that purified UCHL3S75E displayed the enhancing cleavage activity towards Ub-AMC compared to UCHL3WT by 70%, but no physiological level activity to di-ubiquitin (diub) chains, which suggested that the mechanism by which UCHL3 cuts ubiquitin chains might be more complex. Meanwhile, sequence alignment and phylogenetic trees analysis showed that the phosphorylated Ser75 only existed in UCHL3 but not in other UCH family members, indicating that the phosphorylation regulation based on Ser75 probably was unique to UCHL3. In addition, UCHL3 was highly conserved in various eukaryotic organisms, implying that the phosphorylation regulation mechanism of UCHL3 was highly conserved in evolution. The results of this research expanded the understanding of the phosphorylation regulation of UCHL3 and laid a foundation for further understanding of its physiological role.

Key words: UCHL3, phosphorylation regulation, ubiquitin chains cleavage activity, DNA damage repair

摘要： 泛素羧基末端水解酶L3（ubiquitin C-terminal hydrolase-L3，UCHL3）是真核细胞泛素羧基末端水解酶家族（ubiquitin C-terminal hydrolases，UCHs）的重要成员，参与了DNA损伤修复等过程。近期研究表明，UCHL3不仅在体外条件下可以切割C端用香豆素修饰的泛素分子（C-terminal conjugate of ubiquitin with 7-amino-4-methylcoumarin，Ub-AMC），当UCHL3上第75位的丝氨酸（Ser75）发生磷酸化后，其在细胞内切割多聚泛素链的活性也明显增强，但这种磷酸化调控尚缺乏体外证据支持。基于此，利用点突变及多种层析技术制备了野生型UCHL3（UCHL3WT）和模拟磷酸化的UCHL3蛋白（UCHL3S75E），在体外生化水平上研究了磷酸化对UCHL3的泛素链切割活性的影响。体外泛素切割实验显示，与UCHL3WT相比，UCHL3S75E切割Ub-AMC的活性提高了70%，但仍不能展现生理水平上的二泛素（di-ubiquitin，diub）切割活性，暗示UCHL3切割泛素链的机制更为复杂。同时，系统发育树与序列对比分析显示，发生磷酸化的Ser75仅存在于UCHL3中，在其他UCH家族成员中并不保守，表明基于Ser75的磷酸化调控是UCHL3所特有的。此外，UCHL3在众多真核生物中高度保守，暗示着该蛋白的磷酸化调控机制在进化上的保守性。研究结果拓展了对UCHL3磷酸化修饰调控的认识，为深入研究其生理角色奠定了基础。

关键词: UCHL3, 磷酸化调控, 泛素切割活性, DNA损伤修复