In Vitro Construction And Characterization Of Ubiquitination And Ufmylation

Post-translational modification(PTM)of proteins,a key step in protein biosynthesis and function regulation,plays an important role in numerous biological processes,and ubiquitination is one of the most important modifications.Substrates modified by ubiquitination usually have two fates: one is degraded by the 26 S proteasome and the other is modified by non degradative ubiquitination that has been implicated in processes such as cell cycle regulation,chromosome inactivation,protein trafficking,tumorigenesis,achondroplasia and neurological disorders.The ubiquitination of substrates require the involvement of ubiquitin cascade enzymes(ubiquitin-activating enzymes,ubiquitinconjugating enzymes,ubiquitin-protein ligases).Under the catalysis of specific ubiquitinprotein ligases,ubiquitin or ubiquitin chains are connected to specific substrates to perform related functions.UFM1 is a newly identified ubiquitin-like protein with its own system for substrate modification.Recent studies have found that UFMylation plays an important role in endoplasmic reticulum homeostasis regulation and DNA damage repair pathway,and the defects of UFMylation system will lead to many cancers.The UFMylation of substrates also require the involvement of related cascade enzymes.Under the catalysis of the E3 ligase complex(UFL1/DDRGK1),the substrates can achieve the modification of single or multiple UFM1 molecules.In this project,the related modified enzymes of ubiquitination and UFMylation system were successfully purified by using the E.coli expression system,and the in vitro ubiquitination and UFMylation reaction system was successfully constructed by using the self purified cascade enzyme.For the reactions involving three different types of ubiquitin ligases,three different methods were used to characterize the modification results: 1)for the modification reactions with strong ubiquitination activity,SDS-PAGE gel electrophoresis can be directly used for separation,and then Coomassie brilliant blue staining can be used for observation;2)For the modification reaction with weak ubiquitination activity,fluorescence labeling technology combined with SDS-PAGE gel electrophoresis is needed to effectively characterize the modification results;3)For the modification reaction in which the molecular weight of substrate is much smaller than that of ubiquitin molecule,Bis-Tris gel electrophoresis was used to separate and then observed by Coomassie brilliant blue staining.For the weak in vitro UFMylation reaction,we used the characterization method of ubiquitination modification results for reference,and used fluorescence labeling technology combined with SDS-PAGE gel electrophoresis to characterize the results of UFMylation of DDRGK1 and p53 proteins.Subsequently,the modification activity of the whole UFMylation system was significantly improved by truncation of the UFM1 conjugating enzyme UFC1 protein.The in vitro reaction system of ubiquitination and UFMylation established in this study has realized the effective characterization of the modification results on the basis of saving costs and improving the ease of operation.Using the purified modified enzymes and related proteins as well as the constructed reaction system,a reaction kit for detecting ubiquitination or ubiquitination modification can be developed.