Investigation Of Bap31 On The Expression Of Proteins Related With Postsynaptic Density

B cell receptor associated protein 31(Bap31)is a trans-membrane protein located on the endoplasmic reticulum(ER),and it is highly expressed in a variety of tissues and organs,such as bone marrow,thymus,brain,heart and so on.It's noted that Bap31,as a carrier protein,is involved in transporting a variety of membrane proteins from the ER to the Golgi,and it is also participated in the endoplasmic reticulum-associated protein degradation(ERAD)pathway.In addition,the fragment of Bap31(p20)cleaved by caspase-8 has functions in the apoptosis process.Moreover,patients with Bap31 mutations exhibit the central nervous system(CNS)diseases symptoms,such as behavioral abnormalities,mental retardation,and neurological deafness.Studies have shown that postsynaptic density(PSD)is an important region of neurotransmission,where abnormal expression of proteins induces CNS diseases.However,the functions of Bap31 in regulating PSD related proteins expression are still unknown.This study was divided into two parts to investigate the molecular mechanism of Bap31 on the PSD related proteins expression.In the first part,shRNA-Bap31 and siRNA-Bap31 were used to construct Bap31 stably knockdown(shRNA-Bap31)and transiently knockdown(siRNA-Bap31)N2a cells,respectively.Real-time PCR was used to detect the mRNA levels of PSD related proteins in shRNA-Bap31 and siRNA-Bap31 transfected cells.There were 13 genes significantly regulated by Bap31,contained with 4 upregulated and 9 downregulated genes.Finally,valosin containing protein(VCP)was selected as the primary target gene.Western blot and Real-time PCR analyses showed that Bap31 regulated the expression levels of VCP both in vivo and in vitro.Moreover,the hyper-unstable mutant of cystic fibrosis transmembrane conductance regulator(CFTR?F508)was used to further investigate the function of Bap31 and VCP in the degradation of CFTR?F508 process.Immunoprecipitation and Western blot analyses indicated that Bap31 regulated the expression levels of VCP and caused the differences in binding quantities between CFTR?F508 and VCP.In CFTR?F508 stable cells changed Bap31 expression and then negatively perturbed VCP expression,immunofluorescence,flow cytometry and Western blot analyses showed that VCP changed the degradation of CFTR?F508 which Bap31 targeted.To further investigate the mechanism of Bap31 on the expression of VCP,we used the immunoprecipitation technique to detect the association between them.The results demonstrated that there was no direct binding between them.However,Real-time PCR and Western blot analyses showed that Bap31 regulated the mRNA and protein levels of E74-like factor 2(Elf2),a transcription factor of VCP.We over-expressed or knocked down Elf2 in Bap31 perturbed cells,the results showed that the promoter activity of VCP contained with Elf2 binding sites,the mRNA and protein levels of VCP were significantly positively regulated by Elf2.It suggests that Bap31 regulates the expression of VCP possibly through Elf2.In the second part,the isobaric tags for relative and absolute quantitation(iTRAQ)technique was used to screen the Bap31 regulated proteins.The results showed that a total of 2989 proteins were identified and quantified in shRNA-Bap31 transfected cells,including 276 upregulated and 228 downregulated proteins.Then,the 504 differential proteins were analyzed and clustered based on gene ontology(GO),protein location,protein functions and kyoto encyclopedia of genes and genomes(KEGG)metabolic pathways.A total of 21 PSD related proteins(9 upregulated and 12 downregulated proteins)were obtained,which genes are located on the X chromosome.One of the regulated proteins was monoamine oxidase A(MAOA).Flow cytometry and Western blot analyses showed that Bap31 negatively regulated the expression levels of MAOA.In order to investigate the molecular mechanism by which Bap31 regulated MAOA expression,we detected the regulation of Bap31 on the protein degradation.The results showed that Bap31 did not significantly affect the protein degradation of MAOA.Moreover,the results of PCR and sequencing showed that the variable number of tandem repeats(VNTR)upstream of MAOA promoter was not affected by knockdown of Bap31.However,Bap31 regulated the expression levels of cell division cycle associated 7 like(R1/RAM2/CDCA7L/JPO2,a transcriptional inhibitor of MAOA).In addition,the knockdown or overexpression of R1 in Bap31 perturbed cells negatively regulated the mRNA and protein levels of MAOA.The results suggest that Bap31 may regulate the expression of MAOA by R1.In summary,this study showed that Bap31 regulated the expression of VCP by Elf2 and participated in the VCP-mediated degradation of CFTR?F508.Meanwhile,Bap31 negatively regulates the expression levels of MAOA,and its mechanism may be dependent on the regulation of Bap31 on R1,a transcriptional inhibitor of MAOA.The above results demonstrated the molecular mechanism of Bap31 on PSD related proteins expression,and lay a theoretical foundation for finding targets to the treatment of CNS diseases.