关键词: UCHL3, 磷酸化调控, 泛素切割活性, DNA损伤修复

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