BmCBP Protein Catalyzes The Acetylation Of Nutritional Storage Protein BmApoLp-Ⅲ And Regulates Its Stability In Silkworm,Bombyx Mori

Acetylation modification is one of the common post-translational modifications(PTM)of proteins.It refers to the transfer and addition of acetyl groups to the protein lysine residue or N-terminal under the catalysis by acetyltransferase.The modification of adding acetyl groups to lysine residues is called lysine acetylation modification.It is a dynamic modification process and is reversible.It is catalytically regulated by lysine acetyltransferase(KATs)and lysine deacetyltransferase(KDACs),and plays an important role in regulating protein function,chromatin structure and gene expression.c AMP responsive element binding protein(CREB)binding protein(CBP)not only can regulate gene transcription,but also has acetyltransferase activity.CBP protein is involved in regulating cell growth,development,and differentiation,as well as affecting tumor genesis.Bombyx mori Apolipophorin-Ⅲ(BmApoLp-Ⅲ)is a lipid-bound hemolymph protein that participates in flight-related lipid transport in insect.In previous studies,it was found that there were a large number of proteins that could be acetylated in silkworm hemolymph proteins by mass spectrometry,including Apolipophorin(Apo Lp),storage protein(SP),and 30 K proteins.In this article,we studied that BmCBP catalyzes the acetylation of BmApoLp-Ⅲ Protein and regulates its stability in silkworm,Bombyx mori.It is helpful to study the mechanism of regulating nutrition storage and hydrolysis utilization of storage proteins by BmCBP and the acetylation in the silkworm,Bombyx mori.Firstly,we prepared the polyclonal antibody against BmApoLp-III.The BmApoLp-III gene(Gen Bank accession number: NM＿001043613.2)was obtained from NCBI and its open reading frame was then analyzed using bioinformatics software.We used Primer 5.0 to design primers and amplified the ORF of BmApoLp-Ⅲ by PCR technology.Then,through a series of molecular cloning experiments,the recombinant prokaryotic expression vector p ET-28a(+)-BmApoLp-Ⅲ was constructed.The recombinant plasmid was transferred into E.coli BL21(DE3),and the expression was induced by IPTG.The expressed bacterial was collected,ultrasonically broken,and purified BmApoLp-Ⅲ target protein was obtained by nickel column affinity chromatography.In the end,the purified target protein was used to immunize rabbit to prepare anti-BmApoLp-Ⅲ polyclonal antibody.The antibody titer and specificity were detected by ELISA and Western Blotting,respectively.The results showed that the antibody titer could reach 1:1024000,and Western Blotting also detected specific band of BmApoLp-Ⅲ in Bm N cells,suggesting that we have successfully prepared polyclonal antibody against BmApoLp-III with high specificity and titer.Secondly,eukaryotic expression and acetylation identification of BmApoLp-III protein were performed.we linked the ORF fragment of the BmApoLp-Ⅲ gene amplified by PCR technology to the p IEx-1-EGFP vector to construct a transient expression vector p IEx-1-EGFP-BmApoLp-Ⅲ.The plasmid was extracted and transfected into Bm N cells for over-expression.Wild cells were used as control.Anti-His antibody is used to detect the over-expressed BmApoLp-III protein.At the same time,the BmApoLp-Ⅲ protein was enriched by immunoprecipitation using anti-His antibody,and the protein acetylation was confirmed with Western Blotting using pan-acetylation antibody.The results showed that specific bands responding to over-expressing BmApoLp-Ⅲ protein and acetylation were all detected,indicating that we have successfully expressed BmApoLp-III protein in Bm N cells,and confirmed its acetylation modification.Furthermore,we investigated the effect of acetylation modification on the expression level of BmApoLp-Ⅲ protein through drug treatment experiments.The recombinant plasmid p IEx-1-EGFP-BmApoLp-Ⅲ was transfected into Bm N cells for over-expression.Then,the cells were treated with a mixture of deacetylase inhibitors Cocktail and CBP/p300 acetylase inhibitor A-485,respectively.We found that after treatment with Cocktail,the expression level of BmApoLp-Ⅲ protein increased,while after treatment with A-485,the expression level of BmApoLp-Ⅲ protein showed a downward trend.At the same time,we also used the prepared anti-BmApoLp-Ⅲantibody to detect the endogenous BmApoLp-Ⅲ protein after treatment with drugs.The results showed that after treatment with Cocktail and A-485,the expression level of endogenous BmApoLp-Ⅲ protein was consistent with the above results.Finally,we detected the changes of BmApoLp-Ⅲ m RNA level after treatment of Cocktail and A-485 by q RT-PCR.There was no significant change in the m RNA level of the BmApoLp-Ⅲ gene after drugs treatment.In summary,acetylation modification can affect the expression of BmApoLp-Ⅲ protein at a post-transcriptional level.Finally,we confirmed that BmCBP,as an acetyltransferase could catalyze the acetylation of BmApoLp-Ⅲ protein and regulate its stability.First,we demonstrated the interaction between BmCBP and BmApoLp-Ⅲ protein by Co-Immunoprecipitation.Next,Nano ds RNA was used to knock down the level of BmCBP gene in Bm N cells and the expression level and acetylation level of BmApoLp-Ⅲ protein was then detected using Western Blotting.The results showed that the expression level of BmApoLp-Ⅲ protein decreased,and the acetylation level also decreased,indicating that BmCBP can catalyze the acetylation of BmApoLp-Ⅲ protein.Secondly,the Bm N cells were co-transfected with p IEx-1-EGFP-BmApoLp-Ⅲ plasmid and p IEx-1-Ub plasmid,and then treated with Cocktail,A-485,and Nano ds RNA,respectively to explore the changes in the ubiquitination level of BmApoLp-III protein.It was found that after treatment with Cocktail,the ubiquitination level decreased.At the same time,after treatment with A-485 and Nano ds RNA,the ubiquitination level showed an upward trend,indicating that BmCBP can affect the ubiquitination level of BmApoLp-III protein,thereby regulating protein stability.In summary,BmCBP can catalyze the acetylation of BmApoLp-III protein and regulate its protein stability in silkworm,Bombyx mori.