Seipin mutation at glycosylation sites activates autophagy in transfected cells via abnormal large lipid droplets generation
Seipin mutation at glycosylation sites activates autophagy in transfected cells via abnormal large lipid droplets generation
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糖基化位点的 Seipin 突变通过异常大脂滴的产生激活转染细胞中的自噬
DOI：
10.1038/aps.2014.164
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发表时间：
2015-04-01
影响因子：
8.2
通讯作者：
Wu, Li-jun
中科院分区：
文献类型：
--
作者：
Fan, Hua-dong;Chen, Shao-peng;Wu, Li-jun
目的：Seipin是一种存在于内质网中的蛋白质，参与脂质代谢紊乱和运动神经病。方法：将糖基化位点突变Seipin（N88 S或S90 L）质粒和GFP-LC 3质粒分别转染HEK-293、H1299和MES 23. 5细胞，观察Seipin对细胞自噬系统和脂滴形态的影响。对细胞进行免疫荧光和流式细胞术测定，并对细胞裂解物进行免疫印迹分析。结果：突变Seipin蛋白N88 S或S90 L的过表达激活了3种细胞系的自噬，并显著改变了自噬体标志物GFP-LC 3的亚细胞分布，导致细胞质中出现大量大空泡。GFP-LC 3和突变seipin蛋白的亚细胞定位高度重叠。此外，突变的seipin蛋白导致弥漫性小脂滴融合成更大的脂滴。用自噬抑制剂3-MA（5 mmol/L）处理突变的seipin转染的细胞有利于突变的seipin诱导的大空泡的融合。蛋白质糖基化抑制剂衣霉素可以模拟突变seipin诱导的效应，并且用衣霉素处理野生型seipin转染的细胞（2.5 μ g/mL）产生与突变seipin转染细胞相似的形态学和生物化学性质。seipin糖基化位点的突变破坏了其调节脂滴代谢的功能，自噬作为一种适应性反应来分解异常的脂滴。自噬的中断会加速异常脂滴的融合。
Aim: Seipin is a protein that resides in endoplasmic reticulum, and involved in both lipid metabolic disorders and motor neuropathy. The aim of this study was to investigate the effects of mutant seipin on autophagy system and the morphology of lipid droplets in vitro.Methods: HEK-293, H1299 and MES23.5 cells were transfected with the plasmids of mutated seipin at glycosylation sites (N88S or S90L) and GFP-LC3 plasmids. The cells were subjected to immunofluorescence and flow cytometry assays, and the cell lysates were subjected to immunoblot analysis. Nile Red was used to stain the lipid droplets in the cells.Results: Overexpression of the mutated seipin proteins N88S or S90L activated autophagy in the 3 cell lines, and substantially 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-LC3 and mutated seipin proteins highly overlapped. Moreover, and the mutated seipin proteins caused diffuse small lipid droplets to fuse into larger lipid droplets. Treatment of mutated seipin-transfected cells with the autophagy inhibitor 3-MA (5 mmol/L) facilitated the fusion of mutated seipin-induced large vacuoles. The protein glycosylation inhibitor tunicamycin could mimic the mutated seipin-induced effects, and treatment of the wild-type seipin-transfected cells with tunicamycin (2.5 mu g/mL) produced similar morphological and biochemical properties as in the mutated seipin-transfected cells.Conclusion: The mutation of seipin at glycosylation sites disrupt its function in regulating lipid droplet metabolism, and the autophagy acts as an adaptive response to break down abnormal lipid droplets. The interruption of autophagy would accelerate the fusion of abnormal lipid droplets.