Preparation And Primary Application Of Linear Ubiquitin Chain Probes

Ubiquitination modification is an important post-translational modification of proteins,in which single ubiquitin or ubiquitin chain molecules are attached to the target proteins under the sequential catalysis of ubiquitin activating enzyme E1,ubiquitin conjugating enzyme E2 and ubiquitin ligase E3.In contrast,deubiquitinating enzymes（DUB）catalyze the reverse process of ubiquitination,deubiquitination,in which these modifiers are hydrolyzed away from the target proteins.Ubiquitin is a small protein of 76 amino acids with a molecular weight of 8.5 k Da,whose seven lysine residues and the N-terminal methionine residue can be modified by other ubiquitin molecules,resulting in eight types of ubiquitin chain molecules.Linear ubiquitin chain,also known as M1-type ubiquitin chain,is formed when theαamino group of N-terminal methionine（Met1）is conjugated to theαcarboxyl group of C-terminal glycine at position 76 of another ubiquitin molecule.Our preliminary investigation,has revealed that the recombinant linear ubiquitin chains were able to modify target proteins using whole chains as modifiers,which provided the tools for the revelation of the enzymes involved in these modification processes and the related modification mechanism.In this study,a new strategy invented in our laboratory was applied to prepare linear ubiquitin chain proteins and probes.Their function and applicability were tested through intra-and extra-cellular experiments.In this study,the recombinant proteins for probe preparation were expressed and purified in E.coli expression system.The linear ubiquitin chain probes were successfully prepared using the intein chemistry synthesis technique.These ABPs were fused with Strep Tag II as a reporter tag,which was used to isolate the enzymes captured by these probes;the linear ubiquitin chains consisted of G75A mutant,G76A mutant or wild-type（WT）Ubs were used as recognition elements,to mediate the non-covalent interaction between a probe and an enzyme;bromoethylamine（Br₂）and propargylamine（Prg）were used as reactive groups,which could capture target enzymes through forming covalent bonds with the cysteine active centres of target enzymes.The modification function and stability of these linear ubiquitin chain proteins and probes were characterized through intra-or extra-cellular experiments using HEK293T,Hep G2 and Hela cells.The results showed that,as modifiers,all three types of linear ubiquitin chains ubiquitinated proteins in these three cell lines.Compared with WT chains,G75A mutant chains were the most unstable,while G76A mutant chains were the most stable and could modify more target proteins.These results indicated that G75A mutation destabilized the linkages of ubiquitin chains as well as ubiquitin protein,while G76A mutation enhanced the stability of ubiquitin chains,these results also implied that a ubiquitination process might be accompanied by a reaction,deubiquitination.The extracellular results showed that WT chain probes did not conjugate to deubiquitinating enzyme OTULIN but could be hydrolyzed by OTULIN,indicating that the prepared WT chains were naturally linked and susceptible to the specific hydrolysis of OTULIN;G75A chain probes did not conjugate to OTULIN,and were more unstable compared to other two types of probes;G76A chain probes were not hydrolyzed by OTULIN,and could not conjugate to OTULIN.Combined to intracellular function results,it was suggested that G75A and G76A mutations affected the stability of ubiquitin chains,and OTULIN belongs to the endonuclease.Functional studies revealed that all three types of linear ubiquitin chain probes could target deubiquitinating enzyme UCH-L3 to form covalent adducts,but did not interact with deubiquitinating enzyme USP15,demonstrating that the prepared probes in this study possessed a targeting specificity.The enzyme-capture results showed that WT-type linear ubiquitin chain probes could successfully isolate various intracellular proteins,including E1,E2,E3 and DUB,from lysates of three cell lines,HEK293T,Hela and Hep G2,respectively,but no adducts were detected using the mutant chain probes.These results suggested that two mutations at the non-chiral residues,G75 and G76,of ubiquitin affected the spatial structure and the flexibility of non-chiral conformation of the linkage sites of linear ubiquitin chains,and then impacted the recognition and the interaction between a probe and a target enzyme.Further,mass spectrometry was carried out to identify these proteins isolated by WT-type chain probes.The results showed that tetra-ubiquitin probe successfully captured E1 ubiquitin activating enzyme（UBE1）,E2 conjugating enzymes（UBE2O,UBE2NL）,E3 ligases（HUWE1,PRKN）and deubiquitinating enzyme（UCH-L5）,while di-ubiquitin probe also captured,besides the above enzymes,E1 activating enzyme（UBA6）,E2 conjugating enzymes（UBE2S,UBE2K,UBE2A,UBE2T）,E3 ligases（TRIM4,RNF40,MNAB,UBE3A）and deubiquitinating enzymes（OTUB1,USP5,USP14,USP24,USP9X）.In addition to these enzymes related to ubiquitination processes,other types of proteins were also captured by WT-type chain probes.In summary,this study successfully prepared functional linear ubiquitin chain proteins and probes,the recombinant chains functioned the intracellular modification,and the probes successfully captured the enzymes related to ubiquitination processes and other proteins.Therefore,the current research provided important materials and technical support for the investigation of linear ubiquitination.