| Objective:Autophagy is a highly evolutionary conserved celular self-consumptive pathway from eukaryotes to human.It is a process by which autophagosomes engulf defective matters inside the cel,then degrade in autolysosomes.It provides nucleotides,amino acids,ATP to sustain cell metabolism and survival under starvation conditions.Autophagy can eliminate misfolded proteins and flawed organelles to keep cell quality.To ensure organisms working properly,autophagy must be in a proper and appropriate level.Dysregulation of autophagy is associated with many disease,including immune disease,cancer,neurodegenerative disease.Now,3-MA(3-methylamine)and CQ(Chloroquine)have been used as anti-tumor drugs.A better understanding of autophagy can promote the development of health care,and open a new approach for pharmaprojects and treatment strategies.The conversation of pro-LC3 to LC3-I,LC3-I to LC3-II and the degradation of P62 are important testing index for estimating autophagy flux.During autophagy promotion,the cleavage of pro-LC3 increase,the lipidation of LC3-I increase,the degradation of P62 increase.Autophagy pathway Consists of six main steps: initiation,nucleation,expansion,completion,docking and fusion,degradation and recycling.The expansion of double-menbrance phagophore relates that the cysteine protease Atg4 B cleaves the five amino acids MKLSV of pro-LC3 where generates LC3-I.After the effects of Atg7,Atg3,Atg12-Atg5-Atg16 L,LC3-I conjugate to phosphatidylethanolamine of phagophore to generate LC3-II,thus phagophore expansions to autophagosomes.All of those contribute to the completion of autophagosomes.Nevertheless,the process of inhibition of the cleavage of pro-LC3 and the lipidation of LC3-I is poorly understood.Here our studies aim to focus on the effects of GA6 on autophagy and its mechanism.Content:Our study consists of six parts.1)Is GA6 associated with autophagy? Screening proteins that interact with GA6,then verify those proteins.2)What impact GA6 has on autophagy? Whether dose GA6 promote or suppress autophagy? 3)Dose GA6 regulate the cleavage of LC3? What is the mechanism that involved in? 4)Dose GA6 regulate the lipidation of LC3? Dose it depend on the generation of LC3-I.5)What are the phenotypes of the GA6 KO mice through autophagy induction.After(60)Co irradiation,analyze the association between autophagy and radiosensitivity.Methods:we construct GA6-/-ZR75-1 cels,GA6-/-C57 mice,GA6 knockdown Hela by siRNA which can effectively knock down GA6.Firstly,to detect the association between GA6 and autophagy,we screen the autophagy protein that interact with GA6 by mass spectrometry and fast protein liquid chromatography.Then co-transfection separately with GA6 to do co-immunoprecipitation(Co-IP)analysis.In order to clarify the research focus,we detect the protein which is directly interacted to GA6 by GST pull-down.Secondly,the presence of LC3 puncta are observed through immunofluorescence.The presence of autophagosomes and autolysosomes are observed through transmission electron microscopy.The conversation of LC3-I to LC3-II is observed through western blot.Treatment with CQ or RAPA separately to analyze the step which GA6 play a curial role in,and whether GA6 regulates autophagy through mTOR.Thirdly,we analyze the cleavage and lipidation of LC3.For the cleavage,we construct Myc-LC3-PLA2 to analyze the cleavage of pro-LC3 in vivo.GST-LC3-GFP is used to analyze the cleavage of pro-LC3 through observing changes of green fluorescence in vitro.Then use GST pull-down assay to analyze how GA6 regulate the cleavage of pro-LC3.For the lipidation,we construct GFP-LC3-Hela cels,measure GFP-LC3-II by flow cytometry in vivo.Observe LC3-I stick to PE beads to elucidate GA6 regulates the lipidation of LC3-I in vitro.Lastly,at the level of C57 mice,survival curves after(60)Co irradiation are show to analyze the radiosensitivity.Results:(1)GA6 is directly associated with LC3.FPLC,MS and Co-IP indicate that GA6 is interacted with LC3,GST pull-down shows that LC3 is directly interacted with GA6.Thus,our study focus on LC3.(2)GA6 inhibits autophagy.Immunofluorescence indicates that siGA6 and knockout GA6 induce aggregation of LC3 puncta in cels.Western blot shows that GA6 cause decreased conservation of LC3 to LC3-II,and the difference become more significant in CQD treatment.TEM demonstrates that knockout GA6 causes dramatic elevation of autophagosomes.Thus,we speculate that GA6 block the formation of autophagosomes.(3)GA6 inhibits the cleavage of pro-LC3.Overexpression GA6 induce less LC3-PLA2 being cleaved in vivo.GA6 obstructs the green fluorescence of GST-LC3-GFP fades away in the presence of Atg4 B in vitro.GA6,Atg4 B interact with LC3 at the same region.Taken together,GA6 and Atg4 B competitively bind to LC3 to inhibition the cleavage of pro-LC3.(4)GA6 inhibits the lipidation of LC3-I.Overexpression GA6 decreases the MFI of GFP-LC3-II in vivo.Less LC3-I is stick to PE beads in the presence of GA6 in vitro.(5)Autophagy results in irradiation resistance,and GA6-/-mice are radiation sensitive.After exposure to the same dose(60)Co irradiation,survival curve indicates that the autophagy group mice die slowly compared with the normal group,and GA6-/-mice die quickly compared with the GA6+/+ group.Conclusion:Our studies suggest that GA6 serves as a new crucial protein that inhibits autophagy.GA6 inhibits the cleavage pro-LC3 through directly interacting with LC3,following less LC3-I generated.GA6 also inhibits the lipidation of LC3-I independent on its role in inhibition of the cleavage pro-LC3.These findings reveal that GA6 causes a block in the expansion of phagophore,subsequently dock the completion of autophagosomes... |
