| Lysine acetylation is a conservative post-translational modification process. In this process, an acetyl-group was added to the lysine residues with the catalysis of acetyltransferase. Tumorigenesis is closely linked to histone acetylation and deacetylation. With the development of detection technology, non-histone acetylation attracted more and more attention from recent studies. The study of the non-histone acetylation has become the mainstream research.Recent research also showed that acetylation of lysine protein could compete ubiquitin sites, inhibiting ubiquitin-mediated proteasome degradation pathway, which enhanced the stability of the protein and increased protein content. On the basis of the previous research, we identified acetylation protein of silkworm by using iTRAQ and nano-HPLC/MS/MS. From the identification of the data, we found that there were several acetylation sites in protein Bm30K-3. The main topic of this project was the acetylation impact on stability of Bm30K-3 and its possible molecular mechanism.We obtained and cloned the target gene bombxy mori 30K-3(Bm30K-3) through bioinformatics analysis. Then the full length of Bm30K-3 gene was cloned into p IEx-1 vector, and then this recombinant vector was fused with transferred vector pFastBacHT B, which was recombined to bacmid to obtain recombinant virus ie1-bacmid-Bm30K-3. The fusion protein, His-Bm30K-3, was successfully detected by Western Blotting.We treated BmN cells with inhibitors TSA/C646, after infected with recombinant virus with containing ie1-bacmid-Bm30K-3 in BmN cells, the Bm30K-3 protein was expressed at early time; Through Western Blotting, we found that content of Bm30K-3 protein increased with up-regulation of acetylation, while Bm30K-3 protein decreased with down-regulation of acetylation. Then BmN cells which had been treated with TSA/C646 were further treated with the protein synthesis inhibitor CHX and proteasome inhibitor MG132. By analyzing the degradation curve and accumulation curves, we hypothesized that acetylation regulate the level of protein by affecting ubiquitin. The results of Western Blotting, after Bm30K-3 protein immunoprecipited and detected by lysine-acetylation pan antibody and lysine-ubiquitin pan antibody(IgG was the negative control), proved the previous point. The above results showed that, acetylation of Bm30K-3 protein might competite ubiquitin, inhibiting ubiquitin-mediated proteasome degradation pathway, which enhanced the stability of the protein and increased protein content.As we know, 30 K had anti-apoptosis activity. By using the apoptosis model of BmN cells induced by H2O2, we identified the protective effect of protein on apoptosis induced by H2O2 and the impact of acetylation on the anti-apoptosis activity of Bm30K-3. The results of MTT and Caspase-3/7 kit showed that Bm30K-3 protein could enhance the vitality of BmN cells in a certain extent. With the increasing amount of transfected plasmid, the activity of Bm30K-3’s anti-apoptotic would also improved, which showed Bm30K-3 also had anti-apoptosis activity. The results, which studied the impact of acetylation on the anti-apoptosis activity of Bm30K-3 by using TSA/ C646, showed that anti-apoptotic activity of Bm30K-3 would improve with the up-regulation of acetylation. Otherwise, its activity would decrease. Combined with the results of previous studies, we speculated that it might be resulted from acetylation, which could enhance the stability Bm30K-3 protein and increase its protein content. The topic was the first time to carry out acetylation function of silkworm Bm30K-3 protein and provided the basis to further research on acetylation mechanisms of storage proteins in silkworm. |
PDF Full Text Request