The Regulation Of ?-arrestins In Toll And Jak/Stat Signaling Pathways And The Function Of Fibrinogen-related Proteins In Shrimp Antibacterial Immunity

Immune system in vertebrates is consisted of innate immunity and adaptive immunity,but invertebrates lack of specific adaptive immunity,mainly depend on innate immunity to defend against pathogen invasion.So far,a deep research has been carried out in innate immunity of Drosophila.In Drosophila,there exist three main signaling pathways,Toll,IMD(Immune deficiency)and Jak/Stat(Janus kinase/Signal transducers and activators of transcription)pathways,the function and regulation of these pathways have also been reported.In other invertebrates such as shrimp,although some main components in the signaling pathways were identified,the function and regulation of these pathways have less been reported.In the present paper,we found that ?-arrestins were involved in the regulation of Toll and Jak/Stat pathway.We also studied the function of fibrinogen-related proteins(FREPs)in shrimp antibacterial immunity.1.?-Arrestins negatively regulate the Toll pathway in shrimp by preventing Dorsal translocation and inhibiting Dorsal transcriptional activityToll signaling pathway plays an important role in innate immunity of Drosophila melanogaster and mammals.The activation and termination of Toll signaling are finely regulated in these animals.Although the primary components of Toll pathway were identified in shrimp,the functions and regulation of the pathway are seldom studied.We first demonstrated that Toll signaling pathway plays a central role in host defense against S.aureus by regulating expression of antimicrobial peptides in shrimp.We then found that ?-arrestins negatively regulate Toll signaling in two different ways.P-Arrestins interact with the C-terminal PEST domain of Cactus through the arrestin-N domain,and Cactus interacts with the RHD domain of Dorsal via the ankyrin(ANK)repeats domain,forming a heterotrimeric complex of?-arrestin-Cactus-Dorsal,with Cactus as the bridge.This complex prevents Cactus phosphorylation and degradation,as well as Dorsal translocation into the nucleus,thus inhibiting activation of the Toll signaling pathway.?-Arrestins also interact with non-phosphorylated ERK(extracellular regulated protein kinase)through the arrestin-C domain to inhibit ERK phosphorylation,which affects Dorsal translocation into the nucleLs and phosphorylation of Dorsal at Serine276 that impairs Dorsal transcriptional activity.Our study suggests that ?-arrestins negatively regulate the Toll signaling pathway by preventing Dorsal translocation and inhibiting Dorsal phosphorylation and transcriptional activity.2.?-Arrestin 1's Interaction with TC45 Attenuates Stat signaling by dephosphorylating Stat to inhibit antimicrobial peptide expressionImpaired phosphatase activity leads to the persistent activation of signal transducers and activators of transcription(Stat).In mammals,several Stats are identified and Stat family members are often phosphorylated or dephosphorylated by the same enzymes.To date,only one Stat similar to mammalian Stat5a/b has been found in crustaceans and there have been few studies in Stat signal regulation in crustaceans.Here,we report that ?-arrestinl interacts with TC45(45-kDa form of T cell protein tyrosine phosphatase)in the nucleus to attenuating Stat signaling by promoting dephosphorylation of Stat.Initially,we showed that Stat translocates into the nucleus to induce antimicrobial peptides(AMPs)expression after bacterial infection.?Arrl enters the nucleus of hemocytes and recruits TC45 to form the?arrl-TC45-Stat complex,which dephosphorylates Stat efficiently.The interaction of TC45 with Stat decreased and Stat phosphorylation increased in ?arr1-silenced shrimp(M.japonicus)after challenge with V.anguillarum.PArrl directly interacts with Stat in nucleus and accelerates Stat dephosphorylation by recruiting TC45 after V.anguillarum challenge.Further study showed that ?arrl and TC45 also affect AMPs expression,which is regulated by Stat.Therefore,?arrl and TC45 are involved in the anti-V.anguillarum immune response by regulating Stat activity negatively to decrease AMPs expression in shrimp.3.A fibrinogen-related protein(FREP)is involved in the antibacterial immunity of Marsupenaeus japonicusFibrinogen-related proteins(FREPs)in invertebrates have important functions in innate immunity.In this study,the cDNA of FREP was identified from the kuruma shrimp Marsupenaeus japonicus(MjFREP2).The full-length cDNA of MjFREP2 is 1138 bp with an open reading frame of 954 bp that encodes a 317 amino acid protein comprising a signal peptide and a fibrinogen-like domain.MjFREP2 could be detected in hemocytes,heart,hepatopancreas,gills,stomach,and intestines.MjFREP2 could also be upregulated in hemocytes after V.anguillarum and S.aureus challenge.Agglutination and binding assay results revealed that the recombinant MjFREP2 bound to bacteria and polysaccharides.Immunocytochemical analysis results showed that MjFREP2 proteins were mainly distributed in the cytoplasm of hemocytes from unchallenged shrimp and transported to the membrane or secreted out of the cell after V.anguillarum or S.aureus challenge.The secreted MjFREP2 bound to the bacteria presented in shrimp hemolymph.The overexpression of MjFREP2 could enhance bacterial clearance by inducing the phagocytosis of hemocytes.This ability was impaired by knockdown of MjFREP2 with RNA interference.The cumulative mortality of MjFREP2-silenced shrimp was significantly higher than that of the control shrimp.These results suggested that MjFREP2 has an important function in the antibacterial immunity of M.japonicus.