20-hydroxyecdysone Promotes Cleavage Of Apoptosis-inducing Factor 1 To Cause Mitophagy And Apoptosis

Research background and existing scientific questions:Apoptosis-inducing factor（AIF）plays a dual role in organisms.The full-length expression of AIF is located in the mitochondrial inner membrane and participates in the synthesis of energy in the electron transport chain to maintain mitochondrial homeostasis.The degraded C-terminal fragment enters the nucleus and binds with DN A endonuclease to form apoptotic complex,which binds and cleaves DNA to cause chromosome degradation and apoptosis.However,the upstream factors which regulating AIF fragmentation are poorly studied.Complete metamorphosis insects undergo tissue remodeling during development.The larval tissues undergo autophagy and apoptosis,while the adult tissues proliferate.This process is regulated by the steroid 20-hydroxyecdyone（20E）.It is known that 20E induces autophagy and leads to apoptosis by activating Cysteinyl aspartate specific proteinase（CASP）.However,the regulation of 20E on AIF and its effects on cell fate have not been reported.Moreover,the regulatory mechanism of 20E on mitochondrial homeostasis has not been fully elucidated.In this study,Helicoverpa armigera（H.armigera）,the cotton bollworm,a major agricultural pest,was used as a model to explore the role and molecular mechanism of 20E regulation of AIF in tissue remodeling,and to enrich the knowledge of mitochondrial homeostasis regulation and its biological significance.Research results:1.Bioinformatics method was used to identify three AlFs in the genome database of Helicoverpa armigera:AIF1（XP₀49697456.1）、AIF2（XP₀49697520.1）and AIF3（XP₀21 182458.1）.AIF1 was highly expressed in fat body during metamorphosis.Therefore,it is selected as the research target of this thesis.2.Rabbit polyclonal antibody against AIF1 was acquired.AIF1 expressed full length-AIF1（f-AIF1）in epidermis during feeding stage.Cleaved-AIF1（c-AIF1）was detected in midgut during metamorphosis.In fat body,f-AIF1 expressed during the feeding stage,and c-AIF1 expressed during metamorphosis.3.Tissue immunofluorescence experiments showed that in fat body,AIF1 was localized in the cytoplasm during feeding stage and was translocated to the nucleus during metamorphosis.4.Overexpression of f-AIF1-RFPHis in H.armigera epidermal cell line（HaEpi）showed that 20E induced the degradation of f-AIF1 which localized in mitochondria to c-AIF1 in an autophagy independent manner,induced mitophagy.and c-AIF1 translocation to the nucleus to induce cell apoptosis.5.Because f-AIF1 was only expressed at feeding stage,and cAIF1 was only expressed at metamorphosis stage.Aif was knocked down at feeding stage and metamorphosis stage respectively by RNA interference experiments to study its dual roles.The result shows that f-AIF1 deficiency would lead to insufficient ATP synthesis,developmental retardation,mitophagy and death.c-AIF1 deficiency resulted in decreased apoptosis of fat body cells.6.The same sequence of AIF1 promoter region as Forkhead box transcription factor O（FOXO）binding element（FOXOEB,sequence 5’-TTGTTTAC-3’）was predicted by software,and the results of dual-luciferase reporter showed that 20E induced the increase of AIF1 expression through the downstream transcription factor FOXO.Conclusion:f-AIF1 was located in mitochondria during feeding stage,involved in ATP synthesis,maintained mitochondrial stability,and inhibited autophagy.During metamorphosis stage,20 promoted the expression of Aif1 hrough the transcription factor FOXO,and f-AIF cleaves to c-AIF,leaving mitochondria,leading to mitochondrial instability and mitophagy.c-AIF enters the nucleus and leads to a shift from autophagy to apoptosis.Significance:This study found that steroid hormone 20E can promote Aif transcription and AIF fragmentation,causing mitophagy and apoptosis.It provides new knowledge for the study of autophagy regulation,and provides target genes for pest control.