COP9 Signaling Subunit 5 Monomer Mediates Antimicrobial Immunity By Regulating Hemocytes Proliferation In Eriocheir Sinensis

Hemocytes,are invertebrate immune cells that are similar to blood cells in vertebrates and play a crucial role in innate immunity.Traditionally,mature circulating hemocytes lack the ability to proliferate.However,single-cell RNA sequencing in invertebrates and subsequent functional studies challenge this view.COP9(Constitutive photomorphogenic 9 signalosome,CSN)is a conserved protein complex found in the entire animal community.Among them,COP9 signaling subunit 5(CSN5)is unique and usually exists as a stable monomer,involving many signal transduction pathways,mainly involved in cell cycle regulation,and can participate in the regulation of immune cell homeostasis and immune response by regulating cell cycle.However,the regulation mode of CSN5 on cell proliferation between different species and cells is controversial,and its mechanism is not clear in invertebrates.In view of the fact that the molecular mechanism of the dynamic process of hemocytes proliferation and differentiation in invertebrates is largely unknown,this paper takes Eriocheir sinensis,an important economic species in China,as the research object,and further explores the molecular mechanism of E.sinensis CSN5(EsCSN5)affecting hemocytes proliferation by regulating cell cycle,thereby mediating host antibacterial immunity.In this study,the hemocytes of E.sinensis were infected with Staphylococcus aureus and Vibrio Parahemolyticus respectively,and the proliferation level of hemocytes was detected.Then,the non-proliferation and proliferation hemocytes were collected by flow cytometry,and the gene expression characteristics of the two types of cell subsets were analyzed.The results showed that both Gram-positive and Gramnegative bacteria could induce the proliferation of hemocytes,while EsCyclin E was highly expressed in proliferating cells,and Csn5 was at a low level.In view of the potential function of CSN5 and Cyclin E in the process of hemocytes proliferation induced by bacterial infection in E.sinensis,the complete c DNA sequences of EsCsn5 and EsCyclin E were cloned in this study.The full length of EsCsn5 is 1080 bp,and the open reading frame contains 359 amino acids.The full length of EsCyclin E is 1260 bp,and the open reading frame contains 419 amino acids.Bioinformatics analysis showed that EsCSN5 and EsCyclin E were in accordance with the traditional evolutionary rules.In addition,sequence alignment of amino acids corresponding to domain and threedimensional structure alignment showed that they were highly conserved among species.Real-time quantitative PCR showed that EsCsn5 and EsCyclin E were expressed in different tissues containing hemocytes in crabs.After bacterial challenge,the expression of EsCyclin E was significantly induced,while the expression of EsCsn5 was significantly inhibited,suggesting that the above genes are involved in the regulation of antimicrobial immunity mediated by hemocytes proliferation.Further studies found that when EsCsn5 was knocked down,the proliferation of E.sinensis hemocytes was significantly enhanced,indicating that CSN5 plays a key role in the negative regulation of crab hemocytes proliferation.Subsequent studies have found EsCSN5 distributed in two fractions that including holo-complex and monomeric form,whereas knockdown of EsCsn5 has little impact on the amount of the holocomplex.In order to further improve the regulation mode of EsCSN5 regulating the proliferation-mediated antibacterial immunity of E.sinensis hemocytes,the cell cycle of hemocytes was homogenized to G0 / G1 phase,and the cell cycle of E.sinensis hemocytes was divided into early G1 phase,late G1 phase and S to G2 / M phase by the protein expression level of EsCyclin E at different time points.The cells in these three periods were collected to silence EsCsn5,EsCsn8 and EsCyclin E respectively to detect the effective period of the regulation mechanism.The experimental results show that only knocking down EsCsn5 can significantly accelerate the early process of G1 phase,and only has a significant indigenous effect on this period,suggesting that EsCyclin E is a regulatory path-dependent and potential downstream target.On this basis,the specific molecular mechanism was explored.Overexpression of EsCSN5 and EsCyclin E in HEK293 T cells showed that EsCyclin E was significantly degraded.In vitro ubiquitination test confirmed that this degradation was achieved through ubiquitination modification,and EsCsn5 did not play a role in the transcriptional regulation of EsCyclin E.Co-expression of EsCSN8 and EsCyclin E had no significant effect on EsCyclin E protein.In addition,the survival rate of E.sinensis inhibited by EsCyclin E was significantly decreased,and the concentration of hemolymph bacteria was significantly increased.Therefore,EsCSN5 monomer can be ubiquitinated in early G1 phase to degrade EsCyclin E as a common regulator of blood cell proliferation.In summary,the evidence provided in this study showed that the hemocytes of E.sinensis inhibited the expression of EsCSN5 monomer after bacterial infection,in the EsCyclin E-dependent manner,Cyclin E protein was abundantly enriched in the early stage of G1 phase,and the cell cycle process was accelerated to promote cell proliferation,so as to enhance the bacterial clearance ability and protect the host from infection.In this study,the molecular mechanism of immune cell proliferation in invertebrates,was firstly analyzed,and the role of CSN5 in crustaceans was clarified,which provided an important reference for the improvement of the innate immune mechanism of crustaceans.