The Effect Of Acetylation Modification On The Stability Of Bm30K-15 Protein And Its Mechanism In Bombyx Mori

Acetylation is a highly conservative reversible post-translational modification of proteins.It is widely existed in histones and non-histones and plays an extremely important role in protein function and stability.Bm30 K protein is one of the main components of Bombyx mori hemolymph proteins and participates in many physiological processes of Bombyx mori,such as energy storage,fat transport,embryonic development and immune response.In addition,Bm30 K protein has a certain anti-apoptotic effect on cells.In the early study,the acetylation sites of total proteins were identified in Bombyx mori by acetylated-peptide enrichment technology and nano-HPLC/MS/MS.The results showed that a variety of nutrient storage proteins such as Bm SP2,Bm Apo Lp-III and Bm30K-3 have a large number of acetylation modification sites,and the further studies showed that acetylation modification can regulate the stability of Bm SP2,Bm Apo Lp-III and Bm30K-3.Bm30K-15 protein is a low-molecular-weight apolipoprotein abundant in the hemolymph of Bombyx mori,and acetylation modification was found in this protein with a K98 acetylated site.In the present work,the effect of acetylation on Bm30K-15 protein stability and the mechanism are determined in Bombyx mori.First,the recombinant eukaryotic expression vector p IEX-1-Si-GFP-Bm30K-15 was successfully constructed and the acetylation of the overexpressed Bm30K-15 protein was identified.The recombinant expression vector p IEX-1-Si-GFP-Bm30K-15 was transfected into Bm N cells.Using 6×His and acetylation monoclonal antibodies,the acetylation of Bm30K-15 protein was detected with a high acetylation level by immunoprecipitation(IP)and Western blotting.Secondly,Bm N cells transfected with p IEX-1-Si-GFP-Bm30K-15 were treated with acetylase inhibitor C646 and deacetylase inhibitor Cocktail,LBH589,respectively,and the treated Bm N cells were collected.Results have shown that the expression of Bm30K-15 protein in cells treated with C646 was decreased,while the expression of Bm30K-15 protein in cells treated with Cocktail and LBH589 was increased.The further q RT-PCR result showed that Bm30K-15 m RNA level in Bm N cells showed no significant difference aftertreatment with C646,Cocktail and LBH589,respectively.The above results indicate that the changes in the level of acetylation can affect the expression of Bm30K-15 protein,and the acetylation can increase the protein expression at the post-transcriptional translation level.Finally,the protein synthase inhibitor CHX and the proteasome inhibitor MG132 were used to treat cells that were treated with Cocktail and LBH589 to increase the level of acetylation.Increased cellular accumulation indicated that acetylation can enhance the stability of Bm30K-15 protein.At the same time,acetylation and ubiquitination competition experiment was performed,and the results showed that the up-regulation of acetylation level can reduce its ubiquitination level.Combined with the above results,it was suggested that the acetylation modification of Bm30K-15 protein can compete with its ubiquitination modification,inhibit the ubiquitin-mediated proteasome degradation pathway,and thus improve the stability of Bm30K-15 protein.In addition,the prokaryotic expression vector p ET-28a(+)-Bm30K-15 for expressing Bm30K-15 protein was constructed and transformed into E.coli BL21 to express the Bm30K-15 protein.The recombinant protein was purified and used to immunize the rabbit to obtain a polyclonal antibody against Bm30K-15 protein with high specificity and qualified titer,which laid the foundation for further identification of the function of Bm30K-15 protein in cells.In this study,we found that acetylation can affect the stability of Bm30K-15 protein through the competitive relationship between acetylation and ubiquitination modification,which helps to elucidate the molecular mechanism of nutrition storage and hydrolysis utilization mediated by nutrient storage proteins in silkworm.