title: Seipin mutation at glycosylation sites activates autophagy in transfected cells via abnormal large lipid droplets generation.
abstract: OBJECTIVE
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: 1] School of Nuclear Science and Technology, University of Science and Technology of China, Hefei 230026, China [2] Key Laboratory of Ion Beam Bioengineering, Chinese Academy of Sciences and Anhui Province, Hefei 230031, China.
authors.forename: Hua-dong
authors.initials: HD
authors.lastname: Fan
authors.affiliation: Key Laboratory of Ion Beam Bioengineering, Chinese Academy of Sciences and Anhui Province, Hefei 230031, China.
authors.forename: Shao-peng
authors.initials: SP
authors.lastname: Chen
authors.affiliation: Key Laboratory of Ion Beam Bioengineering, Chinese Academy of Sciences and Anhui Province, Hefei 230031, China.
authors.forename: Yu-xiang
authors.initials: YX
authors.lastname: Sun
authors.affiliation: 1] School of Nuclear Science and Technology, University of Science and Technology of China, Hefei 230026, China [2] Key Laboratory of Ion Beam Bioengineering, Chinese Academy of Sciences and Anhui Province, Hefei 230031, China.
authors.forename: Shao-hai
authors.initials: SH
authors.lastname: Xu
authors.affiliation: 1] School of Nuclear Science and Technology, University of Science and Technology of China, Hefei 230026, China [2] Key Laboratory of Ion Beam Bioengineering, Chinese Academy of Sciences and Anhui Province, Hefei 230031, China.
authors.forename: Li-jun
authors.initials: LJ
authors.lastname: Wu
mesh_terms: D001343:Autophagy; D002460:Cell Line; D044388:GTP-Binding Protein gamma Subunits / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D006031:Glycosylation; D057809:HEK293 Cells; D006801:Humans; D066292:Lipid Droplets / Q000378:metabolism* / Q000648:ultrastructure; D017354:Point Mutation; D015854:Up-Regulation
chemical_list: C432679:BSCL2 protein, human; D044388:GTP-Binding Protein gamma Subunits
references.citation: Curr Opin Cell Biol. 2012 Aug;24(4):509-16
references.pmid: 22726586
references.citation: Hum Mol Genet. 2011 Oct 1;20(19):3831-40
references.pmid: 21750110
references.citation: Nat Genet. 2004 Mar;36(3):271-6
references.pmid: 14981520
references.citation: Trends Mol Med. 2004 Sep;10(9):440-4
references.pmid: 15350896
references.citation: Nat Rev Mol Cell Biol. 2006 May;7(5):373-8
references.pmid: 16550215
references.citation: Cell Death Differ. 2007 Feb;14(2):230-9
references.pmid: 16794605
references.citation: J Cell Biol. 2008 Feb 11;180(3):473-82
references.pmid: 18250201
references.citation: Neurobiol Dis. 2008 Aug;31(2):266-77
references.pmid: 18585921
references.citation: Brain. 2009 Jan;132(Pt 1):8-15
references.pmid: 18790819
references.citation: Biochem Biophys Res Commun. 2009 May 1;382(2):419-23
references.pmid: 19285958
references.citation: Nature. 2009 Apr 30;458(7242):1131-5
references.pmid: 19339967
references.citation: Endocrinology. 2009 Oct;150(10):4552-61
references.pmid: 19574402
references.citation: Cell. 2009 Nov 25;139(5):855-60
references.pmid: 19945371
references.citation: Biochem Biophys Res Commun. 2010 Mar 5;393(2):274-9
references.pmid: 20132792
references.citation: Results Probl Cell Differ. 2010;52:35-46
references.pmid: 20865370
references.citation: Biochemistry. 2010 Dec 21;49(50):10747-55
references.pmid: 21062080
references.citation: PLoS Genet. 2011 Apr;7(4):e1001364
references.pmid: 21533227
references.citation: Trends Endocrinol Metab. 2011 Jun;22(6):234-40
references.pmid: 21419642
references.citation: Trends Endocrinol Metab. 2011 Jun;22(6):204-10
references.pmid: 21497513
references.citation: Hum Mol Genet. 2011 Aug 1;20(15):3022-30
references.pmid: 21551454
references.citation: PLoS Genet. 2011 Jul;7(7):e1002201
references.pmid: 21829381
references.citation: J Lipid Res. 2011 Dec;52(12):2136-47
references.pmid: 21957196