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Seipin mutation at glycosylation sites activates autophagy in transfected cells via abnormal large lipid droplets generation.

Abstract：

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 μg/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.

journal_name

Acta Pharmacol Sin

journal_title

Acta pharmacologica Sinica

authors

Fan HD,Chen SP,Sun YX,Xu SH,Wu LJ

doi

10.1038/aps.2014.164

subject

Has Abstract

pub_date

2015-04-01 00:00:00

pages

497-506

issue

eissn

1671-4083

issn

1745-7254

pii

aps2014164

journal_volume

pub_type

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Seipin mutation at glycosylation sites activates autophagy in transfected cells via abnormal large lipid droplets generation.