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Researches On Inhibitors Of β-N-acetyl-hexosaminidase OfHex1 And Interacting Proteins Of Chitin Synthase Kkv

Posted on:2023-06-07Degree:DoctorType:Dissertation
Country:ChinaCandidate:Y W DuanFull Text:PDF
GTID:1521307031976839Subject:Biochemical Engineering
Abstract/Summary:PDF Full Text Request
Insect pests are serious threats to human health and food sources.The control of insect populations is largely reliant on insecticides.However,the problems faced by pesticides are becoming more and more serious.For instance,the existing insecticides have adverse effects on human health and the ecological environment,and the overuse of these insecticides also causes the problem of insect resistance.One way out of these may be to discover novel insecticidal agents that possess new action modes and targets.Chitin is the main structural component in the exoskeleton,trachea,and peritrophic membrane of insects,but it is absent in plants and vertebrates.The abnormal chitin synthesis or degradation can result in abnormal molting and death in insects.Thus,the key proteins involved in chitin synthesis and degradation are potential targets for developing insecticides.Insectβ-N-acetyl-hexosaminidase(Hex1)is the only chitin hydrolase that can hydrolyze chitooligosaccharides to Glc NAc in molting fluid.Then,the natural products that specifically inhibit the activity of insect Hex1,may achieve green and safe control of pests by disrupting the unique moulting process of insects.Chitin is synthesized by chitin synthase.Studies performed in Saccharomyces cerevisiae showed that next to chitin synthase,chitin synthesis is assisted by several chitin synthase-interacting proteins.However,insect chitin biosynthetic system,in particular its component except chitin synthase,is yet ever known.Therefore,the study of insect chitin synthase interacting proteins may provide not only valuable insight into the understanding of insect chitin biosynthesis mechanism but also new molecular targets for pest control.Our group has conducted in-depth research on the chitin hydrolases involved in the degradation of insect epidermis.We have not only established a mature screening and evaluation system for chitin hydrolase inhibitors,but also successfully obtained the crystal structures of several chitinolytic enzymes,includingβ-N-acetyl-hexosaminidase OfHex1 from agricultural pest Ostrinia furnacalis.These crystal structures provide solid theoretical support for the rational design and improvement of inhibitors against chitinolytic enzymes.Therefore,based on our established system and in-depth understanding of the targets,the inhibitors against OfHex1 were screened,and their inhibition mechanisms were studied in this work.After completing the understanding of insect chitin degradation,the chitin synthesis system has become a new research focus.However,the lack of knowledge on chitin synthesis is particularly evident in insect systems,and it is unclear which proteins might be potential insecticide targets.Therefore,the interacting protein of chitin synthase 1(Kkv)was identified in model organism Drosophila melanogaster,and its physiological function was studied by RNAi.The main results are listed below:(1)Berberine is a highly potent inhibitor of OfHex1The determination of the inhibition constant proves that berberine is a highly potent inhibitor of OfHex1,with a Ki value of 12μM.In addition,the inhibitory activity of berberine and its analogs(thalifendine and palmatine)on other glycosyl hydrolases(GH)is weaker than that of OfHex1,with different degrees of selectivity.Co-crystallization of berberine in complex with OfHex1 and molecular docking indicate that berberine binds with the target enzymes by a noncanonical mode that is not the binding mode of the transition state analog inhibitor or a substrate.The core pharmacophore of berberine is the positively charged conjugate plane,which forms aπ-πstacking interaction with the conserved tryptophan residues in the active pocket,and forms electrostatic interactions with the surrounding negatively charged glutamic or aspartic acid residues.(2)Choline transporter-like protein 2(Ctl2),CG8654,and Kazachoc(Kcc)are Kkv-interacting proteinsCtl2,CG8654,and Kcc were identified as Kkv-interacting proteins in Drosophila melanogaster,via three independent in vitro binding assays:co-immunoprecipitation,split-ubiquitin membrane yeast two-hybrid assay,and pull-down assay.MYTH assays showed that multiple truncations of Ctl2,CG8654,and Kcc interacted with Kkv.MYTH assays also showed that a truncated protein of Kkv that contained the N-terminus,TM1,was necessary but not sufficient for interaction with Ctl2.And the truncated proteins of Kkv,including TM2,TM6,TM7,and TM8 were responsible for the interactions with CG8654 and Kcc.(3)Ctl2,CG8654,and Kcc play important roles in chitin biosynthesisThrough the Gal4/UAS system of Drosophila melanogaster,tissue-specific RNAi for Ctl2,CG8654,and kcc were achieved.Knockdown of Ctl2,CG8654,and kcc in the whole body or chitin-containing tissues(epidermis and trachea)resulted in larval or late pupal lethality,which suggest that Ctl2,CG8654,and Kcc have essential roles in Drosophila melanogaster.With the help of the temperature-sensitive repressor Gal80ts,the knockdown of Ctl2 in the whole wing imaginal disc starts from the second instar.The phenotypes of wings include shrunken and swollen due to accumulated fluid,enhanced surface permeability,decreased chitin content in the wings vein,and the pro-cuticle lost chitin laminar layers accompanied by a narrowed cuticle size.In both kcc-and CG8654-knockdown flies driven by ap-Gal4,whose wings were blistered and exhibited overt changes in shape and size compared to normal wings,varying degrees of decrease in chitin content in the dorsal half of the first longitudinal vein was observed.In CG8654-knockdown wings,the laminar organisation of the procuticle was lost.These phenotypes suggest that Ctl2,CG8654,and Kcc are involved in chitin biosynthesis in wings and are potential targets for developing insecticides.In summary,the study discovered novel and effective inhibitors against the key chitinolytic enzymesβ-N-acetyl-hexosaminidase and revealed their inhibitory mechanism,providing a framework for the development of green insecticides against chitin metabolic enzymes.The study also identified the chitin synthase-interacting proteins Ctl2,CG8654 and Kcc played essential roles in chitin biosynthesis,perhaps providing novel and promising targets for controlling insect pests.
Keywords/Search Tags:Chitin, β-N-acetyl-hexosaminidase, Chitin synthase, Insect growth regulator, Insecticide
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