| Acetylation modification is an important post-translational modification(PTM)of protein in cells,which makes protein functions diversified to meet multiple physiological needs of organisms in the processes of growth and development.Protein acetylation is mainly divided into histone acetylation and non-histone acetylation.Histone acetylation was first discovered in 1964,which mainly involved in cellular DNA repair and chromatin structural transformation.Subsequently,with the rapid development of mass spectrometry,more and more non-histone acetylation sites have been found.CBP protein is an important non-histone acetyltransferase in organisms.Studies have found that mammalian CBP protein participates in the acetylation modification of about two-thirds of non-histone proteins in cells,and plays an important role in life activities,such as various stress responses,cellular energy metabolism,immune response and nerve signal transduction.However,there are currently few studies on the acetylation of non-histone proteins in insects.In this work,the silkworm CBP(Bombyx mori CBP,BmCBP)gene was subcloned and expressed in segments for the first time,which made us have a more in-depth and comprehensive understanding of the function of the BmCBP protein.At the same time,the specific inhibitors and the corresponding binding sites of BmCBP protein were screened,and then we further confirmed that BmCBP can regulate the acetylation modification of the silkworm nutrient storage protein BmApo Lp-Ⅱ.Our study provided a preliminary scientific basis for further elucidating the mechanism of acetylation modification regulating the nutritional storage and hydrolysis utilization of silkworm.First,the open reading frame(ORF)was subcloned based on the predicted BmCBP gene(Gen Bank accession number: XM＿038015130)and the prokaryotic expression and antibody preparation of its antigen region protein BmCBP696 was carried out.After extracting the total RNAs of silkworm,reverse transcribed c DNA was used as the template for PCR amplification of BmCBP gene.The ORF sequence of the predicted BmCBP gene was divided into four segments.The four segments were amplified by PCR using TA cloning method and then inserted to TA vector,respectively.The sequencing results showed that there were many different splicing forms in the ORF region of BmCBP.The antigenicity of the protein encoded by the non-spliced region(1-696 bp region of ORF)was predicted by bioinformatics method,and then the prokaryotic expression vector pET-28a(+)-BmCBP696 of the predicted antigenic protein was constructed,and transformed it into E.coli BL21(DE3).The expression of recombinant BmCBP696 protein was induced by IPTG.The BmCBP696 protein was then purified successfully by nickel column affinity chromatography,immunized New Zealand white rabbits,and the BmCBP antibody with high specificity was obtained.The titer was determined to be 1:64000.This laid a foundation for further functional research of the BmCBP protein in the future.In order to further study the function of BmCBP protein,we then carried out the screening and identification of small molecule inhibitors of BmCBP acetyltransferase activity.First,by using the Autodock molecular docking software,we found that there are spatial match and hydrogen bonding interactions between the small molecule inhibitors A485,C646 and the HAT domain of the BmCBP protein.Then,the BmCBP1152 gene fragment coding BmCBP HAT domain was obtained by bioinformatics analysis.The prokaryotic expression vector pET-28a(+)-BmCBP1152 was constructed,and then the BmCBP1152 protein was expressed and purified.The binding and affinity between the purified BmCBP1152 protein and the drugs A485 and C646 were identified by surface plasmon resonance(SPR).The results showed that the small molecule inhibitors A485 and C646 had strong interaction with the HAT domain of BmCBP protein,and their k D values are 48 nm and 203.9 nm,respectively,indicating that A485 and BmCBP have stronger affinity.Further,we carried out the experimental verification of A485 and C646 inhibiting the activity of BmCBP acetyltransferase.A485 and C646 were added into BmN cells with a certain concentration gradient,respectively,and the acetylation level of histone H3K27,a known substrate protein of BmCBP in the cells was detected by Western Blot.We found that after adding the drugs A485 and C646,the acetylation level of H3K27 showed a significant downward trend,among which the downward trend of A485 was more obvious,which was consistent with the results of SPR experiment.At the same time,after transfecting ds RNA and siRNA of the BmCBP gene into the cells,it was also found that the acetylation level of H3K27 protein in the cells was down-regulated.Then we found through real-time fluorescent quantitative PCR that the ds RNA and siRNA affect the acetylation modification of histone H3K27 by knocking down the m RNA level of the BmCBP gene.The above results showed that A485 and C646 can inhibit the acetyltransferase activity of BmCBP protein by binding to the HAT domain of it.Finally,we carried out the study on the regulation of BmCBP to acetylation modification of silkworm nutrient storage protein BmApo Lp-Ⅱ.First,BmN cells were co-transfected with ds RNA/siRNA and p IEX-1-si-GFP-BmApo Lp-Ⅱ plasmid.The overexpressed BmApo Lp-Ⅱ protein was immunoprecipitated using His monoclonal antibody and the Western Blot using acetylation antibody showed that both ds RNA and siRNA of BmCBP gene could down-regulate the acetylation level of BmApo Lp-Ⅱ protein,indicating that BmCBP may catalyze the acetylation modification of BmApo Lp-Ⅱ.At the same time,we found that the expression of BmApo Lp-Ⅱ protein in BmN cells cotransfected with ds RNA/siRNA was significantly down-regulated.Furthermore,a certain concentration gradient of A485 was added into BmN cells without plasmid transfection or BmN cells transfected with plasmid p IEX-1-si-GFP-BmApo Lp-Ⅱ.The expressions of endogenous and overexpressed BmApo Lp-Ⅱ protein in the cells after adding the drug were detected by Western blot using the previously prepared BmApo Lp-Ⅱ antibody and His antibody,respectively.The results showed that with the increase of the concentration of A485,the expression levels of the endogenous and overexpressed BmApo Lp-Ⅱ protein were significantly down-regulated.The above results indicated that the silkworm acetyltransferase BmCBP can catalyze the acetylation modification of BmApo Lp-Ⅱ and down-regulate its protein expression.This will lay a foundation for further study on the mechanism of BmCBP protein regulating the acetylation function of silkworm nutrient storage proteins and the nutritional storage and hydrolysis utilization of silkworm. |
