Effects Of P62 And Its Interacting Protein Vps34 On Autophagy Of Mouse Macrophages Induced By LPS

The self-degradation of cell components with dysfunction is named as autophagy,which play an important role in maintaining the homeostasis of cells.As a junctin,p62 protein not only could regulate the occurrence of autophagy,but also could be degraded by autophagy.Furthermore,p62 protein mediates multiple signaling pathways through interacting with other proteins in cells.Studying on the biofunction of p62 protein has a great significance for exploring the mechanism of autophagy and signal transduction in cells.In present study,the subcellular localization of p62 protein was analyzed.The macrophages of mice were treated by Rapamycin which was the specific inhibitor of mTORC1-mediated signaling pathway,and the regulation of mTORC1-mediated signaling pathway in the expression of p62 as analyzed.To explore the regulation of mTORC2-mediated signaling pathway in the expression of p62,the shRNA vector of Rictor,a characteristic component of mTORC2-mediated signaling pathway,was constituted and transfected into the macrophages of mice.p62 was overexpressed in the macrophages of mice,and the activity of mTORC1/2-mediated signaling pathway were measured by Western blot.The proteins interacting with p62 protein were selected using co-immunoprecipitation assay and were analyzed by mass spectrometry and Western blot.To study the role of p62 protein in the lipopolysaccharide-induced autophagy,the macrophages of mice were treated by lipopolysaccharide,and the formation of Autophagosome and the change of Beclin-1 protein and LC3 protein were measured.The target proteins interacting with p62 protein were analyzed to study the effects of these target proteins on lipopolysaccharide-induced autophagy.Based on this study,we obtained the following conclusion: 1)p62 protein located not only in endoplasmic reticulum,but also in golgi body;2)The reults of mass spectrometry and Western blot shown that p62 protein could interact with Vps34 and FKBP38 proteins.The analysis of protein interaction network shown that Rictor protein interacted with p62 protein and FKBP38 proteins.These data suggested that mTORC2-mediated signaling pathway may closely relate to p62 protein;3)When the mTORC1 protein and mTORC2 protein were inhibited,the expression levels of p62 and p62 protein was significantly reduced(p<0.05).Overexpression of p62 in the macrophages of mice improved the activities of mTORC1/2-mediated signaling pathway.Similarly,silence of p62 in the macrophages of mice inhibited the activities of mTORC1/2-mediated signaling pathway.These results demonstrated that there is a feedback regulation between mTORC1/2-mediated signaling pathway and p62;4)In lipopolysaccharide-induced autophagy,overexpression of p62 in the macrophages of mice improved the levels of Beclin-1 and LC3 proteins,while silence of p62 in the macrophages of mice inhibited the levels of Beclin-1 and LC3 proteins;5)In lipopolysaccharide-induced autophagy,overexpression of Vps34 in the macrophages of mice improved the levels of Beclin-1 and LC3 proteins.Overall,the present study indicated that p62 protein could locate in the endoplasmic reticulum and golgi body,which is a new localization of p62 in the endometrial system.mTORC1/2-mediated signaling pathway could regulate the expression of p62 and p62 protein also could positive regulate the activities of mTORC1/2-mediated signaling pathway,suggesting that there is a feedback regulation between mTORC1/2-mediated signaling pathway and p62 protein.The analysis of protein interaction network shown that 188 proteins may directly or indirectly interact with p62 protein.p62 and Vps34 proteins all could promote lipopolysaccharide-induced autophagy.This study has analyzed the subcellular localization of p62 protein,explored the regulatory function between p62 protein and mTORC1/2-mediated signaling pathways,and studied the role of p62 protein played in lipopolysaccharide-induced autophagy.The present study have layed a foundation for further research on the biofunction of p62 protein in lipopolysaccharide-induced autophagy.