Ubiquitination And Deubiquitination Of PRAME Protein

ObjectPreferentially expressed antigen in melanoma(PRAME)involved in multiple cellular processes such as cell proliferation,immune responses and cellular signalling pathway.PRAME is expressed in many types of cancers,closely related with much diease progression such as breast cancer,melanoma,haematological malignancies and so on.In recently years,many researches have reported the function of PRAME,but the regulation of PRAME is not clear.Therefore,in this article we firstly reveal the polyubiquitinational modification of PRAME.Furthermore we will discribe the degradation pathway of PRAME and which deubiquitylating enzymes(DUB)can regulate the expression level of PRAME when it overexpressed,and we aim to declear the impotant regulation of PRAME with ubiquitilation and deubiquitilation.Methods1.Huh7、MCF7 cell and other cell types were partly treated with the proteasome inhibitor MG132 and Cullin inhibitor MLN4929,after 6 hours cell lysates were prepared and Western blot analysis were performed to detect the level of PRAME.2.MCF7 and A375 cell were partly treated with Cycloheximide(CHX),after a period of time cell lysates were harvested to detect the level of PRAME by Western blot.3.293 T cell transfected with co-overexpressing of PRAME gene and UB gene,for detecting the ubiquitination level of PRAME;furthermore we constructed the domaindeficient mutant of PRAME and explored the ubiquitination level of them.4.In order to detect the interaction between PRAME and Cullin families,each member of Cullins partly transfected 293 T cell with PRAME gene,cell lysates were harvested for co-immunoprecipitate.And we generated the VHL-box domain-deficient mutant of PRAME(△VHL),then test that weather △VHL interact with Cullins.5.We generated RBX expression plasmid(RBX1 and RBX2)who universally existing in Cullin-RING-E3 ligase enzyme complex,and detected the interaction between PRAME and RBX protein.6.We want to discuss whether PRAME will effect the activation of Cullins.7.We constracted sh RNA of Cullins,then MCF7 cell infected with Cullin’s sh RNA and harvested for Western blot to detect protein level of PRAME.8.MCF7 cell partly infected with 96 DUBs and prepared for Western blot in order to screen the DUB which can regulate the stability of PRAME.9.We reveal USP51 can upregulat the protein level of PRAME,then we generated the enzymatic mutant of USP51(USP51-mut)and infected in MCF7 cell to detected the protein level and half life period of PRAME.10.We contransfected PRAME gene and USP51 gene into 29 T cell to detect the interaction of each other.11.Through co-transfecting USP51/USP51-mut with UB plasmid and PRAME,we want to confirm the impact on ubiquitination level of PRAME protein.12.MCF7 cell transfected with PRAME,RLTK,and luciferase of different signal pathways,and detected the density of fluorescence,to find out which signal pathway affected by PRAME.13.Through partly transfected PRAME or USP51 with RLTK,luciferase into MCF7 cell,we hope to declare whether PRAME or USP51 will influence the amino acid deprivation response and heat shock response.Result1.MG132 and MLN4924 will upregulate PRAME protein level.2.In vivo,PRAME protein degradated through proteasome system.3.The polyubiquitinational modification depended on rhe N-terminal region and LRR domains of PRAME protein.4.PRAME protein interacts with Cullins but PRAME interact with CUL2 rather than other members of Cullin family depend on VHL-box domain.5.PRAME protein interacts with RBX who universally exist in Cullin-RING-E3 ligase complex.6.PRAME protein will not disturb the activation of Cullins.7.Singlely knock down Cullin will not upregulate the protein level of PRAME.8.Through screening DUB expression library,we defined USP51 upregulates the protein level of PRAME.9.USP51 rather than USP51-mut upregulates PRAME and prolongs the half life period of PRAME.10.In 293 T cell,PRAME intercats with USP51.11.USP51 rather than USP51-mut delete polyubiquitin chain of PRAME.12.Through screening signal pathway,we defined PRAME promote amino acid deprivation response and heat shock response signal pathways.13.The regulation of USP51 to amino acid deprivation response and heat shock response signal pathways is same as PRAME.ConclusionPRAME protein modified with polyubiquitination and degradated through ubiquitin-protesome system,and the ubiquitination process rely on the N-terminal and LRR domains of PRAME.PRAME interact with Cullin-RING-E3 ligase complex.We defined USP51 can remove the ployubiquitin chain of PRAME depend on the deubiquitination enzyme activity,thus regulate the protein stability.PRAME and USP51 promote amino acid deprivation response and heat shock response signal pathways.