去泛素酶复合体Ubp3/Bre5的制备及与Cdc48作用（英文）

Preparation of full-length deubiquitinating complex Ubp3 / Bre5 and characterization of interaction with Cdc48

泛素化是一种存在于真核细胞中与生理功能密切相关的蛋白修饰,泛素化与去泛素化处于动态调节过程中.Ubp3是与人USP10同源的酵母去泛素化酶,结合辅引子Bre5在细胞内发挥广泛作用.为研究该复合体的工作机制,制备重组蛋白复合体,在大肠杆菌中成功表达并纯化重组Ubp3与Bre5单体及Ubp3/Bre5复合体,首次成功大规模制备重组Ubp3/Bre5复合体.通过一系列pulldown实验,检验Ubp3/Bre5与AAA家族中泛素选择性ATP酶Cdc48的相互作用模式,结果发现,Ubp3及Bre5无法单独与Cdc48结合,但Ubp3/Bre5复合体可以有效与Cdc48相互作用.提出了Ubp3/Bre5-Cdc48相互作用的新模式,制备了高质量重组Ubp3/Bre5复合体.该研究为通过生化及结构生物学进行分子机制探索奠定了基础.

Ubiquitination modification is a dynamic process essential for eukaryotic cell physiology. Ubp3, the Saccharomyces cerevisiae homologue of human deubiquitinase USP10, together with its cofactor Bre5, plays an active role in numerous cellular processes. Although Bre5 is essential for Ubp3 function in vivo, unfortunately, due to diffi-culty in preparing critical quantities of intact functional Ubp3 and Ubp3/Bre5 reconstitute, systemic characterization on this complex is lacking. Hence, how exactly Bre5 regulates Ubp3 activity still remains elusive. To fill this gap, we report the successful expression and purification of recombinant Ubp3 and Bre5 in Escherichia coli in monomeric and complex form. To our knowledge, this is the first report the successful preparation of full-length Ubp3/Bre5 pro-tein complex in large scale, which allows us to obtain further understanding of molecular bases. The stoichiometric interaction between purified Ubp3 and Bre5 confirmed proper folding of these proteins. To assess the proposed direct interactions between Ubp3 and Bre5 with the ubiquitin selective ATPase associated with a variety of cellular activities （AAA ATPase） Cdc48, series of pull-down assays are performed;results reveal that, neither Ubp3 nor Bre5 alone is able to bind Cdc48. However, the Ubp3/Bre5 complex could bind Cdc48 efficiently, which provids novel insight on Ubp3/Bre5-Cdc48 interaction mode. In summary, our results lay the foundation for future mechanistic evaluation by both biochemical and structural means.