The Effect Of Acetylation On The Stability Of BmApoLp-? Protein And Its Mechanism

Acetylation modification is a highly conservative and reversible post-translational modification of proteins,which occurs widely in various physiological activities of organisms.More and more studies have shown that protein acetylation can participate in a variety of life activities,and plays an important role in the regulation of protein stability and function.We previously performed a high-throughput acetylation identification in the nutrient storage proteins in Bombyx mori hemolymph.It was found that,as an orthologue of human Apolipoprotein protein,BmApoLp-?(Bombyx mori Apolipophorin-?)in Bombyx mori hemolymph has a high level of acetylation modification.In this study,the effects of acetylation on the stability of BmApoLp-?I protein and its mechanism were systematically studied,further elucidating the new mechanism that acetylation modification may participate in regulating the nutrition storage and utilization in silkworm,Bombyx mori.First,the eukaryotic expression of BmApoLp-? in BmN cell and the identification of its acetylation were carried out.The total RNAs of the silkworm were obtained from the Bombyx mori ovary epithelial cell line(BmN)by the Trizol method,and then reverse transcribed into cDNA.The BmApoLp-? ORF was amplified by PCR,and then ligated to our constructed eukaryotic expression vector pIEX-1-si-GFP.Using this vector,GFP protein could be co-expressed with the target protein.The co-expressed GFP protein can be used to determine the transfection efficiency and also serve as a control protein in the following experiments.The recombinant vector pIEX-1-si-GFP-BmApoLp-? was transfected into BmN cells.After 48h of transfection,BmApoLp-? was expressed successfully by Western blotting using 6×His monoclonal antibody.At the same time,immunoprecipitation(IP)by 6×His monoclonal antibody was used to isolate BmApoLp-? protein.The western blotting using acetylation monoclonal antibody showed that BmApoLp-? protein had higher acetylation modification.In conclusion,we successfully expressed BmApoLp-?protein in BmN cells,and the protein was highly acetylated.Then,the effect of acetylation level on the expression of BmApoLp-? protein was studied.The deacetylase inhibitor Cocktail and acetylase inhibitor C646 were used to treat the BmN cells transfected with pIEX-1-si-GFP-BmApoLp-? plasmid to up-regulate and down-regulate the level of acetylation,respectively.Compared with the co-expressed control protein GFP,the up-regulation of acetylation level by Cocktail increased the expression level of BmApoLp-? fusion protein,while the down-regulation of acetylation level by C646 resulted in a significant decrease in the level of BmApoLp-? fusion protein.There was a positive correlation between the increase or decrease of protein expression and the dosage of drugs used.At the same time,we expressed BmApoLp-? prokaryotically and successfully prepared rabbit polyclonal antibody against BmApoLp-?.The western blotting by rabbit polyclonal antibody showed that up-regulation and down-regulation of acetylation levels also led to up-regulation and down-regulation of endogenous BmApoLp-? protein in BmN cells,as well as the overexpressed BmApoLp-? fusion protein in BmN cells,further suggesting that acetylation can up-regulate the protein expression and cell content of BmApoLp-? in BmN cells.In addition,qRT-PCR assay showed that the change of acetylation level induced by drug treatment could not change the level of mRNA of BmApoLp-? gene,which indicated that acetylation affected the expression and content of BmApoLp-? protein in cells at a post-translational level.Finally,the mechanism of acetylated modification to modulate the expression of BmApoLp-? protein at the post-translational level was studied.Cycloheximide CHX and proteasome inhibitor MG 132 were used to treat cells to detect the protein stability of BmApoLp-? in BmN cells after acetylation.The results showed that when CHX was used to block protein synthesis,the stability of BmApoLp-? protein treated with Cocktail was significantly higher than that of the control group.Similarly,using MG 132 to block the degradation of proteins,Cocktail treatment can further enhance the stability of BmApoLp-? protein and cell content,indicating that acetylation modification can indeed enhance the stability of BmApoLp-? in BmN cells.In order to identify the molecular mechanism of enhancing the stability of the protein by acetylation,the competitive experiment of acetylation and ubiquitylation were carried out.The results showed that when the acetylation level of BmApoLp-? was increased,the level of ubiquitylation decreased.However,when the acetylation level of BmApoLp-? was decreased by C646,the ubiquitylation level increased,indicating that there was a competitive relationship between the acetylation and ubiquitylation modification of BmApoLp-?.According to above results,acetylation might inhibit ubiquitin-mediated proteasome degradation through competing ubiquitylation modification,thus enhancing the stability and intracellular content of BmApoLp-?proteinIn previous studies,high acetylation modification was found in nutrient-storage proteins of the hemolymph in Bombyx mori,which suggested that the acetylation modification of nutrition-storage proteins might be involved in the nutrition storage and utilization of Bombyx mori.The above results lay a foundation for further elucidating the mechanism of acetylation modification to regulate the nutrient storage and utilization of Bombyx mori.