The Deubiquitinating Enzyme USP38 Promotes Cell Proliferation By Stabilizing The Protein Level Of Survivin

ObjectiveSurvivin is the smallest member of the inhibitor of apoptosis(IAPs)family,and it is also one of the components of the Chromosomal Passenger Complex(CPC),which plays an important role in the regulation of cell survival and mitosis.A large number of studies have found that Survivin is closely related to the occurrence and development of tumors.The current research has found that several E3 ubiquitin ligases can ubiquitin Survivin and degrade it through a ubiquitin-proteasome-dependent pathway,thus affecting the downstream biological function mediated by Survivin protein,But whether there is a deubiquitinating enzyme as opposed to ubiquitin ligase that can stabilize Survivin and regulate its biological functions requires further research.In order to answer this question,the study used a gene expression library containing 96 deubiquitinating enzymes constructed in our laboratory to screen out deubiquitinating enzymes that can stabilize the protein level of Survivin,further explore the molecular mechanism of deubiquitinating enzyme regulating the stability of Survivin protein and its biological function in tumorigenesis and development.Methods1.The Survivin expression plasmid was transfected into MCF7 cells.and proteasome inhibitor MG132 was added 6h before sampling,the cell samples were collected and the protein was extracted.The protein level of Survivin was detected by Western Blot.2.The 96 plasmids from the deubiquitinase expression library were co-transfected with Survivin plasmid into MCF7 cells,respectively.36 hours later,the cell samples were collected and the protein was extracted.The protein level of Survivin was detected by Western Blot.3.Quantitative Real-time PCR(q PCR)was used to detect the effect of USP38(Ubiquitination-Specific Protease 38)on the m RNA expression of Survivin.4.Determined the effect of USP38 on the half-life of Survivin by adding protein synthesis inhibitor Cycloheximide(CHX).5.Co-immunoprecipitation was used to determine the interaction between USP38 and Survivin.6.Through deubiquitination experiment to determine whether USP38 deubiquitined Survivin.7.In USP38 Drosophila homologous gene DUBAI and Survivin Drosophila homologous gene Deterin,the relationship between DUBAI and Deterin was determined by half-life test,co-immunoprecipitation and deubiquitination experiment.8.The interaction and regulation between USP38 and other components of CPC were determined by co-immunoprecipitation and half-life experiment9.To determine the effect of USP38 on the proliferation of MCF7 cells by measuring cell viability by Cell-Counting Kit-8(CCK8).Results1.Compared with the control group,the experimental group added MG132 stabilized the protein level of Survivin.2.When using the deubiquitinating enzyme expression library for screening,it was found that USP38 significantly stabilized the endogenous and exogenous protein levels of Survivin without affecting its m RNA expression level,and can significantly extend the half-life of Survivin.3.Co-immunoprecipitation experiment confirmed the interaction between USP38 and Survivin.4.Deubiquitination experiment confirmed that USP38 deubiquitinated Survivin and depended on its deubiquitinating enzyme activity.5.Co-immunoprecipitation and deubiquitination experiment confirmed that Drosophila homologue gene DUBAI stabilized the protein expression level of Deterin by deubiquitinating Deterin,and there was an interaction between them.6.There was an interaction between USP38 and other components of CPC: Aurora B and CDCA8,and USP38 can extend the half-life of both.7.Cell viability assay confirmed that USP38 promoted the proliferation of MCF7 cells,and knocking down Survivin can counteract this proliferation-promoting phenomenon.ConclusionsWe found through screening that the deubiquitinating enzyme USP38 can stabilize the protein level of Survivin;USP38 interacts with Survivin and USP38 deubiquitinates Survivin by relying on its deubiquitinating enzyme activity.In the Drosophila genome,DUBAI interacts with Deterin and deubiquitinates Deterin to stabilize the protein level of Deterin,indicating that the regulation of survivin protein stability by USP38 is evolutionarily conserved.USP38 can also interact with other CPC components,Aurora B and CDCA8,and stabilize their protein levels.USP38 can also promote cell proliferation by regulating the stability of Survivin protein.