Structure-Function Study Of Scorpion Toxin BmBKTx1 Study Of Proprotein Convertase Furin

This dissertation consists of three major parts.Part 1:bifunctional faces investigation of scorpion toxin BmBKTx1;part 2:adaptor protein mint3 regulates the trans-Golgi network localization of proprotein convertase furin;part 3:design of peptide inhibitors for furin based on the C-terminal fragment of histone H1.2.1.Bifunctional faces investigation of scorpion toxin BmBKTx1Scorpions have survived more than 350 million years with no detectable changes in there anatomy due to their efficient,dynamic machinery production of bioactive peptides mainly blocking Na⁺ channels and K⁺ channels.As one of the most fundamental proteins in organisms,K⁺ channels play key roles in many crucial physiological processes.Till now.scorpion peptides have been proven to be powerful tools for testing the pharmacological,physiological,structural and functional characteristics of K⁺ channels.BmBKTx1 is a novel short chain toxin purified from the venom of the Asian scorpion Buthus martensi Karsch.It is composed of 31 residues and three disulfide bridges.It is structurally related to small conductance Ca²⁺-activated K⁺ channels(SK) toxins,but it only partially inhibited SK currents. The experiment results suggested that BmBKTx1 could specifically block big conductance Ca²⁺-activated K⁺(BK) channels,not voltage-gated K⁺ channels(Kv). As the first reported scorpion toxin active on both BK and SK,BmBKTx1 is insect specific and it was the first reported scorpion toxin active on the Drosophila Slo channel(dSlo).As the first identified scorpion toxin active on both BK and SK channels, BmBKTx1 has been proposed to have two separate functional faces for two targets. To investigate this hypothesis,two double mutants,K21A-Y30A and R9A-K11A, together with wild-type toxin were expressed in E.coli.The recombinant toxins were tested on cockroach BK(pSlo) and rat SK2 channel for functional assay.The mutant K21A-Y30A has a dramatic loss of function on BK but retains its function on SK. The mutant R9A-K11A has no defect on BK and SK.These data supported the two functional-face hypothesis and indicated that the BK face is on the C-terminalβ-sheet. These results can promote the application and development of therapy of BK channel diseases,screening for K⁺ channel toxins and insecticide. 2.Interaction of mint3 with furin regulates the localization of furin in the trans-Golgi networkAfter biosynthesis,peptides and proteins are often activated by intracellular limited proteolysis by proprotein convertases(PCs).These PCs become key regulators in many physiologically and pathologically important processes.Furin is the first identified mammalian PC and the most extensively studied member of the known PCs.Furin play important roles in various biological events such as the activation and secretion of peptide and protein hormones,the maturation of membrane receptors and the activation of plasma proteins.Furin is also involved in many diseases such as the activation of viral glycoproteins and bacterial exotoxins and the metastasis of cancer.Though a few associated proteins of furin were identified and extensively studied in the past years,whether other binding proteins exist and what roles they play in the regulation of furin localization and maturation are still unclear. Searching for and functional investigation of endogenous inhibitors and partners of furin is not only helpful to undestand the function of furin in vivo,but also to develop anti-viral and anti-bacterial drugs.Furin cycles between the plasma membrane,endosomes and the trans-Golgi network (TGN),maintaining a predominant distribution in the latter.Mint3,a member of the Mint protein family,is involved in the signaling and trafficking of membrane proteins.Until now,little has been known about the roles of mint3 in the localization or trafficking of Furin.Here,using co-immunoprecipitation and immunofluorescence assays,we show that mint3 interacts with Furin in the Golgi compartment of HeLa cells.Knockdown of endogenous Mint3 expression by RNA interference disrupts the TGN-specific localization of Furin and increases its distribution in endosomes.We further demonstrate that the phosphotyrosine-binding(PTB) domain of mint3 is essential for the binding of Furin and that this binding affects the TGN-specific localization of Furin.Moreover, mutation studies of Furin indicate that Mint3 regulates Furin distribution mainly through interaction with the acidic peptide signal of Furin.Collectively,these data suggest that the interaction between the PTB domain of mint3 and the acidic peptide signal of Furin regulates the specific localization of Furin in the TGN.These results clarify a new transport and localization pathway of furin;provide a new therapeutic target of furin related disease. 3.Design of peptide inhibitors for furin based on the C-terminal fragment of histone H1.2The mammalian proprotein convertase furin has been found to play an important role in diverse physiological and pathological events,such as the activation of viral glycoproteins and bacterial exotoxins.Small,non-toxic and highly active,furin inhibitors are considered to be attractive drug candidates for diseases caused by virus and bacteria.In this study,a series of peptide inhibitors were designed and synthesized based on the C-terminal fragment of histone H1.2,which has an inhibitory effect on furin.Replacing the reactive site of inhibitors with the consensus substrate recognition sequence of furin has been found to increase inhibitory activity,greatly.The most potent inhibitor-I₄,with 14 amino acid residues- has a K_i value of 17 nM for furin.Although most of the synthesized peptides were temporary inhibitors,the inhibitor I₅,with nine amino acids,retained its full potency,even after a 3 h incubation period with furin at 37℃.These inhibitors may potentially lead to the development of anti-viral and anti-bacterial drug compounds.