The Generation Of Tace Mutant By CRISPR/Cas9 And Functional Analysis In Drosophila

Tumor necrosis factor alpha converting enzyme(TACE/ADAM17)is a metalloproteinase,involved in a variety of extracellular domain shedding.It plays a role in TNFa cleavage and the release of many proteins,such as TGFa,p75 TNFR,L-selectin and?-amyloid precursor proteins.TACE is known to play important roles in tumorigenesis,cell-cell interactions and neurological development.It has been reported that maturation of TACE requires the participation of iRhom2 in mammals;While in Drosophila iRhom is involved in the regulation of epidermal growth factor(EGF)receptor signaling,and iRhom mutant flies exhibit narcolepsy-like phenotype.Based on these information,we come up with hypothesis that the maturation of TACE may also require the participation of iRhom in Drosophila and iRhom-TACE pathway may be involved in the sleep regulation.In this study,we initially tested the molecular interactions between iRhom and TACE in Drosophila by using western blot and co-immunoprecipitation assay.To further investigate the function of TACE in Drosophila,CRISPR/Cas9 system was utilized to generate tace knockout fly strains.Furthermore,sleep analysis was conducted between tace mutants and wild-type flies.Our study ultimately confirms that iRhom regulates TACE maturation in Drosophila melanogaster to modulate sleep,which shed new light on understanding conserved mechanisms regulating this important physiological event both in invertebrates and vertebrates.