Molecular Mechanism Of FBXO45 Degrading USP49 By Ubiquitination In Pancreatic Cancer Cells

Background: Pancreatic cancer is a malignant tumor of digestive tract,which develops rapidly and has a very low survival rate 5 years after diagnosis.Studies have shown that dysregulation of protein degradation mediated by F-box protein can lead to the development of human malignant tumors.Fbxo45 is a member of the F-box protein family that promotes the development and development of a variety of malignant tumors.But the role of FBXO45 in pancreatic cancer has not been reported.Other studies have shown that USP49 can enhance the efficacy of gemcitabine and inhibit the proliferation of pancreatic cancer cells,but the specific mechanism of action is not clear.Our previous study found that FBXO45 promoted the development of pancreatic cancer,while USP49 inhibited the development of pancreatic cancer.After FBXO45 expression was knocked down in PATU-8988 pancreatic cancer cells,USP49 protein expression level was significantly increased.Therefore,this study will further explore the specific molecular mechanism of FBXO45 regulation of USP49.Purposes: To investigate whether FBXO45 can down-regulate USP49 through ubiquitination and the detailed molecular mechanism;To investigate whether FBXO45 affects the development of pancreatic cancer cells by down-regulating USP49.Methods: After overexpression or downregulation of FBXO45 in pancreatic cancer cells,qRT-PCR and Western blot were used to detect whether the mRNA and protein expressions of USP49 were changed.The protein half-life of USP49 was determined by actinomycin assay after the increase or decrease of FBXO45.After MG132 proteasome inhibitor was added to pancreatic cancer cells,FBXO45 was detected to down-regulate USP49 protein expression through ubiquitin proteasome pathway.The specific binding of FBXO45 to USP49 was determined by CO-IP assay.In vitro ubiquitination experiments were conducted to determine whether FBXO45 could enhance the ubiquitination level of USP49.Finally,rescue experiments were conducted to increase the expression of USP49 in pancreatic cancer cells overexpressing FBXO45,or downregulate the expression of USP49 in pancreatic cancer cells knockout FBXO45,so as to detect the changes in the growth,apoptosis,migration,invasion and other malignant biological behaviors of pancreatic cancer cells.Results: 1.After knockdown or overexpression of FBXO45 in pancreatic cancer cells,the expression level of USP49 protein was significantly increased or decreased,but the mRNA level of USP49 did not change.2.Protein half-life experiments showed that down-regulating FBXO45 slowed the rate of decline in USP49 protein levels after treatment with actinomycin(CHX).3.Co-immunoprecipitation showed that FBXO45 could bind specifically to USP49.4.After the deletion of SPRY domain,FBXO45 no longer specifically binds to USP49;After the deletion of the conserved base sequence of USP49,it is no longer regulated by FBXO45.5.After the expression of NEK6 is down-regulated,the level of USP49 protein is significantly increased,and the interaction and degradation of FBXO45 with USP49 can be enhanced.6.Rescue experiments demonstrated that FBXO45 negatively regulates USp49-mediated cell proliferation,migration,and invasion after it is simultaneously increased in FBxo45-overexpressing pancreatic cancer cells or down-regulated in FBxo45-knockout pancreatic cancer cells.Conclusions: Here,we report that USP49 stability is governed by Fbxo45-mediated ubiquitination and is enhanced by the absence of Fbxo45.Moreover,Fbxo45 binds to a short consensus sequence of USP49 through its SPRY domain.Furthermore,Fbxo45-mediated USP49 ubiquitination and degradation are enhanced by NEK6 kinase.Functionally,Fbxo45 increases cell viability and motility capacity by targeting USP49 in pancreatic cancer cells.