Structural Insights into the Phosphorylation-Enhanced Deubiquitinating Activity of UCHL3 and Ubiquitin Chain Cleavage Preference Analysis

Ubiquitin C-terminal hydrolase-L3 (UCHL3), an important member of the ubiquitin C-terminal hydrolase family, is involved in DNA repair and cancer development. UCHL3 can cleave only complexes of monoubiquitin and its conjugates, such as Ub-AMC, His, or small ubiquitin-like modifier, but not polyubiquitin chains. Phosphorylation of Ser75 promotes the cleavage activity of UCHL3 toward poly-ubiquitin chains in vivo, but biochemical evidence in vitro is still lacking. Here, we first analyzed the structure of simulated phosphorylated UCHL3(S75E) and the complex of UCHL3(S75E) with Ub-PA and preliminarily explained the structural mechanism of phosphorylation-enhanced UCHL3 deubiquitinating activity. Additionally, the cleavage activity of UCHL3 toward different types of synthesized poly-ubiquitin chains in vitro was tested. The results showed that purified UCHL3(S75E) enhanced the cleavage activity toward Ub-AMC compared to UCHL3(WT). Meanwhile, UCHL3(S75E) and UCHL3(WT) did not show any cleavage activity for different types of di-ubiquitin and tri-ubiquitin chains. However, UCHL3 could hydrolyze the K48 tetra-ubiquitin chain, providing compelling in vitro evidence confirming previous in vivo results. Thus, this study shows that UCHL3 can hydrolyze and has a cleavage preference for polyubiquitin chains, which expands our understanding of the phosphorylation regulation of UCHL3 and lays a foundation for further elucidation of its physiological role.

Automated summary

This study analyzed the phosphorylation mechanism of UCHL3 in its deubiquitinating activity and provided in vitro evidence of UCHL3's cleavage activity towards specific types of polyubiquitin chains.