Screening Of Deubiquitinating Enzymes That Regulate The Protein Stability Of Themselves

Background:Deubiquitinating enzymes(DUBs)are a large class of enzymes in the ubiquitin system.DUBs remove ubiquitin molecules from ubiquitinated proteins to prevent protein degradation and regulate intracellular localization,thereby maintaining protein homeostasis and playing important roles in regulating the function of target proteins.Interestingly,DUBs are also regulated by post-translational modifications(including ubiquitination).However,whether DUBs can reverse themself ubiquitination of themselves still need further investigated.Objective:The purpose of our work is to use the gene expression library of deubiquitinating enzymes and their enzymatic mutations to find out whether some DUBs that can regulate the stability of their own protein levels.Moreover,we will study mechanism of how DUBs regulated protein stability of themselves.Method:1.Design cloning primers for cloning of DUBs enzymatic mutants,use PCR technology to amplify the target genes,and finally establish the gene expression library of DUBs enzymatic mutations.2.Transfected the DUBs and their enzymatic mutants plasmids into MCF7 cells using liposome EL transfection reagent.After 40-48 hours,the cell was harvested,and the protein expression levels of indicated genes were detected by Western-blot.3.The enzymatic mutants of the DUBs gene selected above were co-transfected with corresponding wild-type DUBs or control plasmids into MCF7 cells by transfection reagent EL,then the cell was harvested,and the proteins were detected by Western-blot.We found that 11 DUBs up-regulated the protein expression level of the corresponding enzymatic mutants.4.Among the candidate genes found above,a conserved USP subfamily including USP26,USP29 and USP37 attracted our attention.We will focus on the mechanism of how these USP subfamily members regulated protein stabilities,including whether these DUBs interacted with themselves and promoted selfdeubiquitinated.5.Take USP29 as an example to study the domains that regulate the stability of USP29 protein and the mechanism by which USP29 regulates the protein stability of itself.Results:1.Successfully constructed DUBs enzymatic mutant gene expression library which has 47 DUBs.2.After screening of the gene expression library of DUBs and their enzymatic mutants,we found that 13 genes with active DUBs mutations that led to a decrease in their protein levels were selected.3.11 DUBs are capable to regulate their protein stability.4.USP26,USP29,and USP37 subfamily underwent ubiquitin-dependent degradation in MCF7 cells.5.The USP37 subfamily members regulated protein stabilities of their enzymatic mutant through homologous self-interactions.6.USP26,USP29 and USP37 underwent self-deubiquitinated.7.USP26,USP29 and USP37 extended the protein half-life of their enzymatic mutants.8.The N-terminal domain of USP29 protein is unstable.9.USP29 stabilized N-terminal domain of USP29 protein.10.USP29 interacted with its N-terminal domain,and the N-terminal domain underwent homo-interaction.11.The N-terminal domain of USP29 undergoes ubiquitin-dependent degradation in MCF7 cells.12.The N-terminal domain of USP29 protein underwent K27 type polyubiquitination.Conculsion:We screened DUBs and their enzymatic mutants expression libraries(47 genes)and found that some DUBs regulate the stability of their own proteins through homointeraction in an enzymatic activity dependent manner.Taking USP29 as an example,we found that the self-deubiquitination of USP29 inhibited the degradation of its own enzymatic mutants and protect it from the ubiquitin-proteasome degradation system.Our work indicated that the N-terminal region of USP29 is a key domain for regulation of protein stability of USP29 protein.The N-terminal region of USP29 is modified with K27-type ubiquitin.In summary,our work reveals that some DUBs regulate self-ubiquitination and their protein stabilization.Our work provides novel molecular insights into the diverse regulatory mechanisms of deubiquitinase.