The Study On The Stability Of BmApoLp-? Regulated By Acetylation Modification In Bombyx Mori

Apolipophorin-III?ApoLp-??is a nutrition-storage protein that can transports fat in the hemolymph of insects.It belongs to the apolipoprotein family and is synthesized in the fatty body.It is mainly used in the storage and transport of lipid,and also plays an important role in the immunoreaction,inflammation and resistance to the infection of external bacteria.Acetylation modification is an important reversible protein post-translational modification?PTM?that regulates the interaction between protein and DNA,gene transcription,stress response,metabolism and protein stability,and also related with many important diseases such as diabetes,cancer and cardiovascular diseases.Our previous studies have shown that there are a large number of acetylated sites in various nutrition-storage proteins including Apolipophorin-??BmApoLp-??in the hemolymph of Bombyx mori,in which the stability of SP2 and 30K-3proteins can be regulated by acetylation modification.Based on the previous studies,the function of acetylation modification to regulate the stability of Bombyx mori BmApoLp-?was also studied,which laid a foundation for further study on the mechanism of acetylation in regulating the storage and utilization of nutrition in silkworm.Firstly,the ORF fragment of BmApoLp-?gene was obtained from the total RNAs of silkworm ovary cells by RT-PCR and cloned into the eukaryotic expression vector pIEX-1-Si-GFP to obtain the recombinant eukaryotic expression vector pIEX-1-Si-GFP-ApoLp-?.The HIS-fused recombinant protein BmApoLp-?was successfully expressed in silkworm BmN cells.The purified BmApoLp-?protein was obtained by immunoprecipitation with HIS monoclonal antibody,and the acetylation monoclonal antibody was used to detect acetylation by Western blotting.The results showed that BmApoLp-?was highly acetylated,which was consistent with the previous identification by mass spectrometry.Secondly,we up-regulated and down-regulated the acetylation level by LBH589 and C646,respectively,and the expression level of Bm ApoLp-?protein was also up-regulated and down-regulated,respectively.The results showed that the lysine acetylation could affect the expression and cell content of BmApoLp-?protein at post-translational level.CHX and MG132 were used to study the effect of acetylation modification on protein degradation and accumulation.The results showed that the up-regulation of acetylation level could significantly slow down the degradation of BmApoLp-?protein after blocking the protein synthesis pathway.Additionally,the up-regulation of acetylation level could further increase the cell content of BmApoLp-?protein after blocking the degradation pathway of BmApoLp-?,which indicated that the up-regulation of acetylation level could improve the stability of Bm ApoLp-?protein.Finally,the competition between acetylation and ubiquitination of Bm ApoLp-?protein was identified.The results showed that when the acetylation level of BmApoLp-?increased,the level of ubiquitination decreased,and vice versa,the levels of acetylation and ubiquitination were negatively correlated.It was proved that the acetylation of BmApoLp-?could compete with its ubiquitination.These results suggested that acetylation modification could up-regulate the expression and stability of BmApoLp-?protein at posttranslational level,and the molecular mechanism may be due to that the acetylation could compete for its ubiquitination,blocking the proteasome degradation pathway mediated by ubiquitination.Thus,the stability and cell content of protein were improved.The above results suggested a new regulation mechanism for the stability and hydrolysis of BmApoLp-?protein cound be found in Bombyx mori.In addition,we used H₂O₂ to induce apoptosis of BmN cells,which proved that BmApoLp-?protein had the ability of anti-apoptosis in a dose-dependent manner.LBH589 treatment could further improve the survival rate of BmN cells,while C646 could significantly decrease the survival rate of cells.The results showed that acetylation could improve the anti-apoptosis ability of Bm ApoLp-?protein.Based on the previous results,it is speculated that the up-regulation of acetylation level could improve the protein stability,thus improving the anti-apoptotic ability of BmApoLp-?protein.