Functional And Mechanism Studies On BmCaspase-8-like In Autophagy

Autophagy refers to the process in which damaged organelles,protein aggregates and invading pathogenic microorganisms are wrapped in a new type of bilayer structure and transported to lysosomes and degraded into macromolecules,and finally reused by cells under pressure conditions.Autophagy participates and regulates many physiological processes,including development,immunity,cancer and aging.Bombyx mori is a lepidoptera model insect of great economic value.Its life cycle includes four stages: egg,larva,pupa and adult.During metamorphosis,some organs in silkworm will be degenerated,and autophagy and apoptosis play an important role in this process.The spatiotemporal expression profiles of Bmcasp8 l,autophagy-related genes and apoptosis-related genes showed that expression of BmCasp8L precedes BmAtg8.Western blotting and confocal microscopy revealed that BmCasp8L inhibits the cleavage of BmAtg6 by BmDREDD and promotes starvation-induced autophagy.In this paper,we systematically studied the function and regulation mechanism of BmCasp8L gene in autophagy of silkworm,and provided a theoretical basis for revealing the regulation mechanism of autophagy of silkworm and other insects.The main research results are as follows:1.BmCasp8L promotes starvation-induced autophagyBmCasp8L consists of two tandem DED domains.It inhibits BmDREDD-mediated apoptosis by forming amyloid aggregates and suppressing its self-activation.The expression profile of Bmcasp8l showed it is highly expressed in the posterior silk glands at the pre-pupal stage,and further analysis revealed that Bmcasp8l and the autophagy marker Bmatg8 had the same expression pattern in the posterior silk glands during the pre-pupal stage to the pupal stage.Autophagy related genes Bmatg5,Bmatg6 and apoptosis related genes Bmdredd,Bmcaspase-1 were all up-regulated to certain degrees from spinning stage to pupation stage.During the degeneration of posterior silk glands and midgut,the protein level of BmCasp8L peaked at the end of the fifth instar,while BmAtg8-PE peaked at the end of spinning stage.After establishing autophagy induction model in BmE cells,we found that BmAtg8-PE was significantly increased when BmCasp8L was overexpressed.Consistently,the protein level of BmAtg8-PE was significantly reduced when BmCasp8L was knocked down by ds RNA.We also found that the number of cells containing BmAtg8-EGFP puncta was significantly increased when BmCasp8L was overexpressed,while it was significantly reduced when BmCasp8L was silenced.Lyso Tracker-Red staining assay further demonstrated that overexpression of BmCasp8L significantly promotes starvation-induced autophagy.2.BmCasp8L promotes autophagy by inhibiting the BmDREDD-mediated cleavage of BmAtg6Autophagy inhibitor Baf A1 effectively inhibited the degradation of BmCasp8L during autophagy,while proteasome inhibitor MG-132 did not,demonstrating that the degradation of BmCasp8L after starvation induction depends on autophagy.Co-localization of BmCasp8L-m Cherry and BmAtg8-EGPF during autophagy further confirmed that BmCasp8L was aggregated in autophagosomes.Co-expression of BmCasp8L and BmAtg6 showed that BmCasp8L significantly inhibited the cleavage of BmAtg6 after starvation induction.Considering BmAtg6 contains the cleavage site of BmDREDD and catalytic activity of BmDREDD could be inhibited by BmCasp8L,we co-expressed BmCasp8L,BmDREDD and BmAtg6,and found that BmDREDD could promote the cleavage of BmAtg6,which is inhibited by BmCasp8L.