Studies On The Regulation Mechanism Of Silkworm Atg8 Acetylation Modification In Cell Autophagy And Apoptosis

Autophagy is a highly conserved process involved in the turnover of intracellular materials in eukaryotes to respond to external stress conditions and promote the survival of living organisms.During this process,some proteins or organelles that need to be degraded are enveloped and transported with a bilayer membrane structure to the lysosome by autophagosomes.There are many Atgs(autophagy-related protein)that normally take part in this process.As one of the two ubiquitin-like proteins,Atg8(autophagy-related 8 protein)in conjugation with PE(phosphatidylethanolamine)is located in the autophagosomes membrane during the autophagy.The modification of the Atg8 changes its behavior,though its molecular mechanism has been studied thoroughly in mammals,its autophagy regulation mechanism in insect cells is rarely reported,especially the regulation by post-translational modification of proteins.As a model organism of Lepidoptera,the complete metamorphic process of Bombyx mori is affected by factors such as nutrition and ecdysone.And these factors can participate in the regulation of autophagy.Thus,it is of great significance to further study the regulatory mechanism of the Atg8 in the autophagic and apoptotic processes of the Bombyx mori.Our previous result of lysine acetylome in silkworm showed that acetylation occurs at the K13 site of Atg8.And acetylation of protein is one of the most important post-translational modifications that plays a very significant role in beings.Consequently,in order to further understand the molecular regulation mechanism of acetylation of the silkworm Atg8 protein in cell autophagy,we used a prokaryotic expression system to express the silkworm Atg8 in bulk and prepared polyclonal antibodies.In addition,the site-directed mutagenesis was thoroughly used to study the molecular mechanism of autophagy regulation by acetylation modification of the protein.Firstly,we extracted the total RNA of the BmN cells and amplified the atg8 gene of the silkworm by RT-PCR.Then prokaryotic expression vector pET28a-atg8 was constructed and recombinant protein was induced and purified.After that,polyclonal antibody was prepared by immunize New Zealand white rabbits.The EBSS(Earle' Balanced Salt Solution)was used to stimulate BmN cells to establish the autophagy model in order to study the effect of starvation on the expression of Atg8 in Bombyx mori.The results showed the possible occurrence of autophagy within a very short time after induction,and also the proportion of Atg8-PE/Atg8 peaks after 2-4 hours of starvation.In the process,the mimicked acetylated and deacetylated mutants were constructed and inserted into the transient expression vector pIEX-1.The BmN cells were then transfected with the recombinant plasmid to overexpress the recombinant protein.WB analysis showed that,the acetylation modification of Atg8 inhibits the occurrence of cell autophagy.Using the fluorescent protein mCherry as a marker with vector p IEX-1,the fusion expression was performed in Bombyx mori cells to observe the difference in autophagy between the cells during,before and after the modification of Atg8.The results showed that,the level of autophagy after Atg8 acetylation was low and it would be higher after Atg8 is deacetylated.Finally,the effects of acetylation modification of Atg8 protein on apoptosis were analyzed by DNA fragmentation detection reagent and cell flow cytometry.The results showed that the rate of apoptosis in BmN cells which contained the acetylated Atg8 protein was higher.In summary,the K13 acetylation modification of autophagy-associated protein Atg8 of the silkworm had an inhibitory effect on the occurrence of autophagy,and therefore its deacetylation would inhibit the occurrence of apoptosis to some extent.This result will lay a foundation for further study of the molecular mechanism of autophagy regulation in silkworm cells.