Autophagy System Is Involved In Seipin Mutation-caused Lipid Metabolic Disorder And α-particles Radiation-induced Bystander Effect

Autophagy is a molecular and cellular process that responsed to inner and outer stress of the cell. In this dissertation, we mainly focused on the role of autophagy when cells met with the inner stress (lipid metabolism-related gene mutation) and the outer stress (a-particle irradiation).The dysfunctional protein caused by gene mutation is a common stress factor for the cells. Seipin is a protein that resides in the endoplasmic reticulum, which mutation at glycosylation site results in protein misfolding and therefore leads to lipid metabolic disorder. Through tranfection experiment in vitro, we aimed to study the effect of autophagy in abnormal morphology of lipid droplets caused by glycosylation site mutation of seipin gene. Cells were transfected with seipin plasmids (glycosylation site-mutated) and autophagosome marker GFP-LC3plasmid. Nile Red was used to monitor cellular lipid droplets, and the cells were subjected to immunofluorescence, immunoblot analysis and flow cytometry assays. We found that overexpression of mutated seipin altered the sub-cellular distribution of the autophagosome marker GFP-LC3, leading to a number of large vacuoles appearing in the cytoplasm. The sub-cellular location of GFP-LC3and the mutated seipin proteins was highly overlapped, and the autophagy system was activated. Moreover, the mutated seipin proteins caused the diffuse small lipid droplets to fuse into larger lipid droplets. Treatment of mutated seipin-transfected cells with the autophagy inhibitor facilitated the fusion of mutated seipin-induced large vacuoles. Interestingly, the protein glycosylation inhibitor could produce the mutated seipin-induced effects, enabling the cells transfected with normal seipin plasmids to display similar morphological and biochemical properties as the mutated seipin-transfected cells. In a summary, gene mutation disrupted the normal morphology and the metabolic function of lipid droplets, whereas the induction of autophagy acted as an adaptive response to break down the abnormal lipid droplets.Radiation is an important physical stress factor outside the cells. Radiation-induced damage has been approved to exsist in non-irradiated cells, which was defined as Radiation-induced bystander effects (RIBEs). It’s significant for radioprotection and radiotherapy to study the mechanism of RIBEs. To date, the relationship between autophagy and RIBEs has been seldom studied. The RIBEs could be mediated through Gap junctional intercellular communication (GJIC). We found that autophagy was induced under the low dose radiation in irradiated and bystander cells, and connexin43, a key molecule of GJIC, was a substrate of autophagy, also, connexin43could mediate the propagation of ROS from irradiated cells to bystander cells. The ROS deriving from irradiated cells was the trigger for inducing autophagy in irradiated and bystander cells. Interestingly, high dose radiation-induced ROS inhibited autophagy, elevating the level of connexin43and further promoting the transmission of ROS from irradiated cells to bystander cells. By using autophagy inducer, the level of ROS decreased significantly in bystander cells, reducing cell death. As a result, it’s conducive to elucidating the mechanism of non-target effects for underlying application in radioprotection and radiotherapy from the aspect of autophagy.