Study On The Mechanism Of NEDDylation Of PML/RAR? And Its Role In The Differentiation Therapy Of Acute Promyeloytic Leukemia

ObjectiveThe pathogenesis of Acute promyelocytic leukemia(APL),a distinct subtype of acute myeloid leukemia(AML),is closely related to the formation of PML/RAR? fusion protein that cause the normal differentiation of myeloid cells blocked in the promyelocytic stage.There are three major subtypes(L,S,V)of PML/RAR? fusion proteins in clinically,of which L and S are the major subtypes,accounting for 95%of the tota.The formation of PML/RARa fusion protein interferes with the growth,differentiation,maturation and apoptosis of normal cells in the following aspects:it changs the localization of PML,interfers with the formation of PML nuclear bodies and inhibits the normal activity of PML protein.All of above result in abnormal proliferation,inhibiting apoptosis and inhibiting the transcriptional activation of RAR?.PML/RAR?homodimer can compete with RARa for the RARE of the retinoid response element so that the transcriptional activation of the target gene regulated by RAR? is inhibited which causing M3 AML.In addition to the occurrence,development and diagnosis of APL,PML/RAR? also plays an important role in the differentiation of APL.At present,ATRA and arsenic trioxide,which are used in the clinical differentiation therapy,both achieve therapeutic effects by degrading PML/RAR? and inducing cell differentiation.Thus,the PML/RARa fusion protein is the basis of the pathogenesis of APL,and the core of the differentiation therapy is degradating of PML/RAR? fusion protein.PML/RAR?degradation pathway mainly includes ubiquitin proteasome pathway,the autophagic lysosome pathway and the caspase pathway.The substrate need to be changed in spatial conformation and subcellular localization before they are degraded to be recognized by different degradation pathways.The post-translational modification of proteins precisely controls these changes.Post-translational modifications of the PML/RAR? fusion proteins include SUMOylation and ubiquitination,both of which are involved in the degradation of PML/RAR? fusion proteins.NEDDylation is a ubiquitin-like,posttranslational modification where the NEDD8(neural precursor cell-expressed,developmentally downregulated 8)molecule is covalently to lysine residues of substrates.Like ubiquitination,NEDDylation involves E1,E2,and E3 enzymes.NEDD8 is highly conserved in most eukaryotes,whereas aberrant NEDDylation can lead to tumors and neurodegenerative diseases,all of which suggest that NEDDylation plays an important role in life activities.Our previous studies found that NEDD8 interacts with PML/RARa and the inhibition of NEDDylation affects the stability of the PML/RARa protein,which have not been reported previously.Thus,we will further investigate the molecular mechanisms involved in the NEDDylation regulation of PML/RAR?,elucidate the effects of NEDDylation on the degradation of PML/RAR?,and explore the possibility of targeting NEDDylation of PML/RARa as novel differentiation strategy.MethodsCOS-7 cells were transfected with PML/RARa plasmid.Interaction of PML/RARa with NEDD8 was detected by immunoprecipitation.Lentivirus-shRNA specific against UBC12 mRNA to silence the UBC12 expression.Western blot detected the PML/RAR?protein levels when treated with NEDDylation inhibitor MLN4924 or silencing UBC12.The effect of NEDDylation inhibitor MLN4924 on nuclear bodies(NBs)formation was detected by immunofluorescence.Cell growth curve was performed by counting cells and trypan blue staining.Cell surface maker CD11b was detected by flow cytometry.Cell differentiation-related biochemical function was evaluated by NBT-reducing activity.Results(1)PML/RAR? interacts with NEDD8.Lysate of COS-7 cells transfected with PML/RAR? plasmid were subjected to immunoprecipitation and western blot.The data revealed that covalent binding can occur between PML/RARa and NEDD8,which was blocked by MLN4924,a specific inhibitor of NEDD8.Correspondingly,COS-7 cells were cotransfected with PML/RAR? and NEDP1(a deNEDDylation enzyme)plasmids.Whole cell lysates were subject to western blot and immunoprecipitation.The result showed that NEDP1 could deNEDDylation of PML/RAR?.UBC12 is a E2 enzyme of NEDDylation.Extracts from COS-7 cells,which were cotransfected with UBC12 and PML/RAR?,were subjected to immunoprecipitation.The data showed that overexpressing UBC12 can significantly enhanced NEDDylation of PML/RAR?.On the basis of this,UBC12 point mutation to UBC12-C111S that make ubc12 lost enzyme activity,the NEDDylation of PML/RARawas inhibited.COS-7 cells were transfected with PML or RARa plasmid.Cell lysate were subjected to immunoprecipitation and western blot.The data showed that RARa instead of PML can occurred NEDDylation.Taken together,these observations showed that PML/RAR? can specifically covalently bind to NEDD8,and the binding site is located in the RARa part of PML/RAR?.(2)Effect of NEDDylation of PML/RARa on Protein Stability.Exponentially growing NB4 cells were treated with different concentrations of MLN4924 for 24 and 48 hrs.Whole cell extracts were subject to western blot analysis.The data showed that PML/RARa fusion protein degraded when MLN4924 treated 48 hrs.Lentivirus-shRNA specific against UBC12 mRNA to silence the UBC12 expression in NB4 cells,and inhibited the NEDDylation of PML/RAR?.Western blot results showed that shUBC12#1 and shUBC12#3 downregulated the expression of PML/RAR?,shUBC12#2 was relatively weaker.The above experiment showed that NEDDylation can stabilize the expression of PML/RARa fusion protein.(3)Effect of NEDDylation of PML/RAR? on NB formation.Hela cells were overexpressed PML/RAR?,and treated with different concentrations of MLN4924(0,0.125,0.25,0.5 ?M)were for 48 hrs.The formation of NB was detected by immunofluorescence.The results showed that when MLN4924 treated with 0.25 ?M and 0.5 ?M after 48 hrs,PML/RARa dispersed into a dot-like aggregation,NB formed obviously.Correspondingly,when treated MLN4924(0.5 ?M)after different time(0,12,24,48 hrs),the formation of NB became obviously.The above experiments showed that inhibiting NEDDylation of PML/RARa promote the formation of NB.(4)Study on the relationship between NEDDylation of PML/RAR? and APL cell differentiation.To further explore effect of inhibiting NEDDylation of PML/RAR?,three shRNA plasmids targeting UBC12 were designed and introduced into APL cells.The cells number and viability were determined by trypan blue eaclusion with cells counting in Burker chambers.The result showed that shUBC12#1 and shUBC12#3 could significantly inhibit the proliferation of NB4 cells.The result of cytological examination of CD11b showed that inhibition of NEDDylation of PML/RAR? could promote the differentiation of APL cells,and the positive rates of CD11b in shUBC12#1 and shUBC1#3 reached 18.3±1.3%and 16.0±5.4%after the fifth day.The experimental results further confirmed that inhibition of NEDDylation can induce NB4 cell differentiation.The above experimental results showed that inhibition of NEDDylation of PML/RARa promote acute promyelocytic leukemia cell differentiation.ConclusionOur study found that PML/RARa fusion protein is a new substrate protein of NEDDylation,which can interact with NEDD8.NEDDylation of PML/RAR? fusion protein increases protein stability.However,the specific inhibitor of NEDDylation MLN4924 or silence shUBC12 down-regulated the expression of PML/RARa protein.Inhibition of NEDDylation of PML/RAR? can significantly promote the formation of NB and APL differentiation.This study not only found new posttranslational modifications of PML/RAR?,but also suggested that the inhibition of NEDDylation of PML/RARa may be a potential new target for the differentiation therapy of acute promyelocytic leukemia.