| Background:Insulin-degrading enzyme(IDE)is a multifunctional zinc-metalloendopeptidases that regulates development.It can not only degrade insulin and glucagon as a protease to regulate cell proliferation and apoptosis,but also participate in a variety of physiological processes through the interaction with other proteins,such as binding to steroid hormone receptors to regulate hormone signaling pathways.Dysfunction of IDE can lead to diabetes and Alzheimer’s disease.It is an important target for drug development.However,the exact mechanism of how IDE alters subcellular localization to combine with different targets to perform different functions remains unclear.Protein post-translational modification(PTM)refers to the covalent attachment of modification groups to proteins,which can quickly regulate protein activity and subcellular localization.Succinylation is one of the acylation modifications on lysine residues.Its covalent group comes from succinic acid,an intermediate product of the tricarboxylic acid cycle.It can change the charge properties of lysine residues and then regulate protein function.It is a new research hotspot of PTM.Therefore,our scientific question is,will the subcellular localization and target selection of IDE be regulated by PTM,especially succinylation?What is the specific mechanism?Metamorphosis is important for insects to adapt to the environment,which not only involves the change of morphology,but also involves programmed cell death of larval tissue and reconstruction of adult tissue.It is also regulated by sterol hormones and insulin.During metamorphosis,the nutritional and energy states of insects change dramatically,which may lead to significant changes in protein PTM.In conclusion,insect metamorphosis is a good model for exploring the regulation of IDE subcellular localization and target selection by PTM.Therefore,this thesis intends to take Helicoverpa armigera as the object to explore the function and mechanism of IDE in insect metamorphosis.The results can improve the mechanism of subcellular localization and upstream regulation of IDE,and then provide reference for drug development and pest control.Results:We cloned and identified the Ide from H.armigera and recombinant expressed it in Escherichia coli.Polyclonal antibodies were prepared by immunizing rabbits.The temporal and spatial expression of IDE were detected by western blot.IDE mainly distributed in fat body and midgut,and total protein levels showed no significant difference between feeding and metamorphic stages.IDE was purified from midgut and fat body by immunoprecipitation.and the changes of PTM were detected by western blot.The results showed that the succinylation of IDE increased significantly during metamorphosis.When Ide was knocked down in metamorphic stage via injecting with dsRNA.the transcription of genes related to the 20E signaling pathway was suppressed,and the larvae failed to pupate and then died.In H.armigera epidermal cell lines.2 μM 20E significantly increased the succinylation level of IDE.After mutation at lysine 179(K 179),IDE succinylation was significantly reduced.Cytochemistry and western blot assay showed that IDE could be translocated from cytoplasm to nucleus after 20E stimulation and that succinylation on K 179 was necessary for this process.Subsequent protein interaction experiments showed that the succinylated IDE could bind to ecdysone receptor EcRB1.Conclusion:Ecdysone 20E can induce succinylation of lysine 179 of IDE during metamorphosis.Succinylated IDE can translocate from cytoplasm into nucleus and bind to ecdysone receptor EcRB1 to promote 20E signaling pathway and promote the metamorphosis of H.armigera.The results provide evidence that hormone regulate the subcellular localization of IDE via PTM and then acts on specific targets. |
