Substrates Verification Of Ubiquitin Ligase E4B And Construction Of USE1 Stable Cell Line

Ubiquitin ligase E4 B is a special enzyme that has both E3 and E4 functions in the ubiquitination system.As E3,it can prolong the polyubiquitin chain of the substrates.As E4,it can promote the polyubiquitination and degradation of p53 mediated by ubiquitin ligase MDM2,thereby promoting survival of cancer cell.E4 B also plays an important role in various biological processes such as embryonic development,DNA damage response and repair,and regulation of nervous system development.Therefore,investigating of the downstream substrates protein of E4 B helps to improve the understanding of E4 B,and also helps to discover the role of ubiquitination in regulating the biological process of substrates protein.In the preliminary results of the laboratory,more than one hundred possible substrates of E4 B in cell were found.This study investigated whether the eight potential substrates NIPSNAP1,DNAJA2,PYCR2,PM20D2,PABPC1,TACO1,KARS,and CORO1 C could be ubiquitinated by E4 B at the protein and cellular levels.Through the detection of ubiquitination and the CHX chase experiment,NIPSNAP1,DNAJA2,and PYCR2 were found to be E4 B ubiquitination substrates.Ubiquitin-binding enzyme USE1 is a specific target enzyme of ubiquitin-activating enzyme UBA6,and does not interact with another ubiquitin-activating enzyme UBA1.Looking for the downstream substrates of USE1 through proteomics can provide strong support for the study of UBA6-USE1-specific ubiquitination pathway.In this paper,the experimental conditions of USE1 overexpression have been established,and sh USE1 stable cell line have been selected,laying the foundation for the verification of downstream substrates of USE1.