Usp7 Regulates Hippo Pathway Through Deubiquitinating The Transcriptional Coactivator Yorkie

The dynamic balance of protein regulation is crucial for maintaining the normal growth and development of organisms.Ubiquitination modification is a widespread post-translational modification of proteins,which refers to the process of covalent coupling of ubiquitin molecules to target proteins under the catalysis of ubiquitin activating enzyme(E1),ubiquitin coupling enzyme(E2)and ubiquitin ligase(E3),modification of target protein specifically.Ubiquitination modification can regulate the stability,localization and activity of target proteins.The ubiquitination modification of protein is a reversible process.Under the action of deubiquitinating enzymes(DUBs),the ubiquitin and poly-ubiquitin chains that bind to the substrate protein are hydrolyzed and removed,resulting in the deubiquitinating modification of the substrate protein.Ubiquitination and deubiquitinating work together to regulate cell DNA damage response,signal transduction,immune response,growth and development,etc.Under the joint regulation of ubiquitin ligase and deubiquitinating enzyme,the organism can reach a stable state.Deubiquitinase regulates organ development and tumorigenesis through a variety of signal pathways.Ubiquitin specific protease 7(Usp7)is one of the most widely studied deubiquitinating enzymes.Up to now,high expression levels of Usp7 have been detected in multiple myeloma,neuroblastoma,prostate cancer,breast cancer,ovarian cancer and other tumors.It can be seen that Usp7 dysfunction is related to tumorigenesis.Therefore,various components in the ubiquitin process are considered as potential targets of treatment strategies for diseases and cancer.Hippo signaling pathway regulates cell proliferation and apoptosis through the co-transcription factor Yorkie(Yki),and plays a key role in organ development,tissue size homeostasis,tissue regeneration,tumorigenesis,wound healing and immune regulation.The upstream of Hippo signaling pathway is mainly the cascade reaction of some kinases such as Hpo and Wts,which can change the subcellular localization of Yki by regulating the phosphorylation level of the co-transcription factor Yki(Yki),thus affecting its activity and signal transmission.When Hippo signaling pathway is activated,Hpo will undergo phosphorylation modification,and then through Wts,phosphorylated Yki,phosphorylated Yki binds with 14-3-3 protein,leaving Yki in the cytoplasm;When the Hippo signalling pathway is deactivated,the non-phosphorylated Yki enters the nucleus and combines with the transcription factor Sd to activate the downstream target gene expression,promote cell proliferation,inhibit cell apoptosis,and promote organ growth.Hippo signaling pathway is highly conserved from Drosophila to mammals.Hippo signaling pathway disorder can lead to many diseases,most notably cancer.Therefore,Hippo signaling pathway has become a target for treatment and intervention of various diseases.However,after the co-transcription factor Yki enters the nucleus,how its function is terminated to maintain a stable state of the organism,however,how Yki protein is regulated is still unknown.In this study,we investigated the protein stability of the co-transcription factor Yki of Hippo signaling pathway,and analyzed its biological function,and acquired the following results:(1)Usp7 promotes organ development and activates the expression of Yki-Sd target geneThrough genetic screening of the deubiquitinizing enzyme in Drosophila,it was found that the knockdown of the deubiquitinizing enzyme usp7 would lead to a smaller wing area,and overexpression of Usp7 could rescue the phenotype caused by the knockdown of usp7.It shows that Usp7 can positively regulate the organ area of Drosophila melanogaster.In recent years,with the continuous deepening of research on Hippo signaling pathway,Hippo signaling pathway is considered to be an important signaling pathway involved in the regulation of organ size,so we speculate that Usp7 may regulate the organ size of Drosophila through Hippo signaling pathway.Since Hippo signaling pathway mainly regulates the expression of target genes through the co-transcription factor Yki,this study examined the effect of Usp7 on the expression of Yki target genes through immunofluorescence staining.The results showed that knockdown of usp7 in the wing disc of resulted in down-regulation of the expression of Yki target genes diap1-lac Z,cyc E and ban-lac Z;Overexpression of Usp7 will up-regulate the expression of Yki target genes.In order to eliminate the off-target effect of RNAi,multiple RNAi lines targeting different regions of usp7 were used in this study.It was found that all lines of usp7-RNAi could inhibit the expression of Yki target genes.In addition,overexpression of Usp7 can also rescue the down-regulated phenotype of Yki target gene caused by knockdown of usp7.At the same time,a phenotype with significantly down-regulated expression of Yki target genes was also detected in the mutant clone of usp7.These results indicate that Usp7 is regulating the expression of Yki target genes.(2)Usp7 regulates Hippo signaling pathway downstream of Wts and upstream of YkiBased on the above experimental results,we propose that Usp7 affects the organ development of Drosophila melanogaster by regulating the activity of Hippo signaling pathway.In order to find the target of Usp7 regulating Hippo signaling pathway,the immunofluorescence staining experiment found that the up-regulated phenotype of Yki target genes cyc E and diap1-lac Z caused by knockdown of hpo or wts could be rescued by knockdown of usp7,indicating that Usp7 was located downstream of Hpo and Wts.The down-regulated phenotype of diap1-lac Z caused by knockdown of yki cannot be rescued by overexpression of Yki.Similarly,the up-regulated phenotype of target gene Cyc E caused by overexpression of Yki cannot be rescued by knockdown of usp7,indicating that Usp7 is located upstream of or parallel to Yki.It is speculated that Usp7 may regulate the protein level of Yki.Through immunofluorescence staining,it is found that the protein level of Yki is significantly reduced in the mutant clone of usp7.Subsequently,the RT-PCR experiment found that knockdown of usp7 inhibited the m RNA level of Yki target genes,and overexpression of Usp7 activated the m RNA level of Yki target genes.The above results indicate that Usp7 can positively regulate the level of Yki.(3)Usp7 interacts with YkiSince the ubiquitinase needs to function by combining with the substrate,we speculate that Usp7 may regulate the activity of Hippo signaling pathway by interacting with a component of Hippo signaling pathway.The interaction between Usp7 and components of Hippo signaling pathway was detected by co-IP experiment.It was found that Usp7 can only bind to Yki,but not to other components of Hippo signaling pathway,and endogenous Usp7 can also bind to endogenous Yki.At the same time,the interaction between Usp7 and Yki was further verified by GST pull down experiment.In order to explore the influence of different domains on the binding of Usp7 and Yki,Usp7 and Yki were segmented and cloned respectively.co-IP experiment showed that the N-terminal of Yki could bind to Usp7,independent of the WW domain of the C-terminal of Yki;Usp7 binds to Yki through the C-terminal,independent of the MATH domain of the N-terminal of Usp7.In addition,this study found that Hpo and Wts of Hippo signaling pathway can inhibit the interaction between Yki and Usp7.(4)Usp7 regulates Hippo signaling pathway depending on its deubiquitinase activityThe above experimental results show that the binding of Usp7 to Yki does not depend on its MATH domain.Therefore,we suspect that the regulation of the expression of Yki target genes by Usp7 may depend on its deubiquitinase activity.Through immunofluorescence staining,it was found that overexpression of Usp7-CA(the form of loss of Usp7 deubiquitinase activity)reduced the level of Yki target genes,indicating that Usp7-CA played a dominant negative role.At the same time,Usp7-CA cannot rescue the phenotype caused by knockdown of usp7,while Usp7-ΔMATH can rescue.In the context of overexpression of Wts,both Usp7 and Usp7-ΔMATH can increase the eye area,but both Usp7-CA and usp7-RNAi decrease the eye area.It is further confirmed that Usp7 regulates the expression of Yki target genes,which depends on its deubiquitinase activity rather than its MATH domain.(5)Usp7 deubiquitinates YkiThe above results suggest that Usp7 may affect the activity of Hippo signaling pathway by deubiquitinized Yki.By detecting the stability of exogenous and endogenous Yki protein,we found that Yki is unstable,and its half-life is about 8 hours.Through the nucleocytoplasmic separation of Yki protein and the use of lysosome and proteasome inhibitors,it was found that Yki in the cytoplasm was mainly degraded by lysosome,and Yki in the nucleus was mainly degraded by proteasome.Western blotting showed that Usp7 could inhibit the degradation of Yki,and knocking down usp7 would accelerate the degradation of Yki.Because Usp7 is located in the nucleus,and Usp7 can stabilize the protein level of Yki.Therefore,it is speculated that Usp7 mainly affects the level of Yki protein in the nucleus.Through nucleocytoplasmic separation and Western blotting,Usp7 mainly stabilizes the Yki of the nucleus and depends on its deubiquitinase activity.The effect of Usp7 on the ubiquitination level of Yki was detected by ubiquitination experiment.It was found that knocking down usp7 would enhance the ubiquitination level of endogenous Yki.Overexpression of Usp7 and Usp7-ΔMATH would inhibit the ubiquitination level of Yki,while Usp7-CA had no effect on the ubiquitination level of Yki.Moreover,Usp7 can significantly deubiquitinating Yki-SA(the phosphorylation deletion form of Yki)rather than Yki-SD(the simulated phosphorylation form of Yki),which indicates that Usp7 can cause the deubiquitinating modification of Yki,which located in the nucleus,and stabilize the protein level of Yki.(6)Usp7 is conserved in Drosophila and mammalsIn order to test the conservation of Usp7 regulating Yki in mammals,the homologous HAUSP of Usp7 was overexpressed in the wing disc.It was found that it increase the level of Yki target genes,and HAUSP could rescue the phenotype caused by knockdown of usp7.In addition,HAUSP can bind to Yki and its homologous YAP in mammals,and endogenous HAUSP can also bind to endogenous YAP.HAUSP can positively regulate the protein level of endogenous YAP.At the same time,HAUSP,the mammalian homolog of Usp7,can also cause the ubiquitination modification of YAP,and then regulate the protein stability of YAP.(7)HAUSP is upregulated in hepatocellular carcinoma(HCC)tissues,and positively correlates with YAPSince the inactivation of Hippo signaling pathway is associated with multiple cancers,and the abnormality of YAP will lead to HCC,so this study uses HCC samples as the research object,and found that HAUSP and YAP are mostly increased in cancer tissues.Through the correlation analysis of the protein content of the up-regulated samples,it was found that there was a positive correlation between HAUSP and YAP.Moreover,overexpression of HAUSP in HCC cell line Huh7 can up-regulate the expression of YAP target genes,knockdown of HAUSP can inhibit the expression of YAP target genes,while HAUSP-CA does not affect the expression of YAP target genes.Through MTT test,it was found that HAUSP could promote the proliferation of Huh7 cells,while HAUSP-CA had no effect on the proliferation of cells.Knocking down HAUSP would inhibit the proliferation of Huh7 cells.In addition,inhibitors of HAUSP can inhibit the expression of YAP target genes and also inhibit the proliferation of Huh7 cells.In summary,this study found that the Hippo signaling pathway can attenuate the interaction between the deubiquitination enzyme Usp7 and the co-transcription factor Yki.At the same time,Usp7 can cause the deubiquitination modification of Yki,thus regulating the Hippo signaling pathway.In addition,the mammalian homologue of Usp7,HAUSP can also make Yki’s homologue YAP in mammalian undergo deubiquitination modification,thus regulating the protein stability of YAP.In addition,this study found that the expression of HAUSP and YAP was positively correlated,and both of them were up-regulated in HCC tissue samples.To sum up,Usp7/HAUSP can stabilize Yki/YAP,and HAUSP can be a potential target for liver cancer treatment.