Functional Research Of TCTP SUMOylation In Silkworm,Bombyx Mori

Translationally regulated tumor protein(TCTP)is expressed in tumor cells at high levels and is a highly conserved protein in plants and animals,which was first found in human breast cancer cells and mouse sarcoma cells.With the continuous research on TCTP,researchers have found that TCTP is involved in many biological process,for example,it can regulate cell cycle,protein synthesis,immune response,anti-apoptotic activity and so on,which plays very important roles in the growth and development of organisms.For a long time,the research of TCTP has never stopped,and the functional mechanism has become clearer.As an important material basis of life activities,protein is affected by many factors.Ubiquitin-like modification,as an important factor,affects protein function by changing protein localization,protein stability,and protein folding.Ubiquitin-like protein,such as SUMO,NEDD8,ISG15,and FUB1,is conjugated to target protein through specific lysines,which is similar to ubiquitin modification.With the development of science and technology in recent years,the understanding of ubiquitin-like modification has been promoted.It can not only affect the function of proteins,but also cause major diseases of the organism.Therefore,researchers have given great concerns on the mechanism of ubiquitin-like modifications.The silkworm is an important economic insect,which is considered as a good model for studying genetics and immunology.However,it has been threatened by Bombyx mori nuclear polyhedrosis virus(BmNPV)for many years in sericulture,causing serious economic losses to the country.It has become an urgent to breed new type of anti-virussilkworm.TCTP is an important immune protein,which has been regulated by various modifications.Therefore,the current study will mainly explore the key sites of SUMOylation and analyze whether and how SUMOylation affects the immune function of Bm TCTP.The following results were obtained from our experiments:1.Functional analysis and identificationin of TCTP SUMOylation in silkwormIn order to identify the key lysine site of BmTCTP SUMOylation,we predicated the lysine site at position 164 of BmTCTP and confirmed it as an important SUMOylation site by Co-IP experiment.Then we constructed cell lines stably expressing wild type of BmTCTP(FLAG-BmTCTP-WT)and its mutation(FLAG-BmTCTP-K164R)and enriched these proteins by immunoprecipitation.Further,we identified the proteins that interact with FLAG-BmTCTP-WT and FLAG-Bm TCTP-K164 R by LC-MS/MS.The result showed that 207 proteins were identified,which were specifically bind to BmTCTP.We found that these proteins are mainly involved in the regulation of protein phosphatase,protein transport,immune response and protein localization in silkworm.To verify whether BmTCTP participates in the immune function,we selected the Bombyx mori nuclear polyhedro virus BmNPV,and infected cells after BmTCTP RNAi for 72 h to detect the replication of BmNPV.The result showed that the replication of BmNPV was continuously increased followed with the decreased expression of BmTCTP by RNA interference.We speculated that BmTCTP may play an important role in inhibiting replication of Bm NPV.2.Effects of TCTP SUMOylation on its function in silkwormSubcellular localization analysis showed that BmSmt3-TCTP,Bm TCTP-WT,and BmTCTP-K164 R were all located in cytoplasm.After infection of BmNPV,lots of BmSmt3-TCTP and BmTCTP-WT proteins entered the nucleus in response to an immune response.Significantly,the SUMOlyated BmTCTP-WT was enriched in the nucleus,whereas only small fraction of BmTCTP-K164 R entered into the nucleus.These results indicated that the SUMOylation could enhance the immune function of BmTCTP by promoting it into the nucleus.Then we detected the replication of BmNPV after RNAi of the endogenous BmTCTP and transfected the same amount of BmSmt3-TCTP,BmTCTP-WT and BmTCTP-K164 R plasmids into BmN cells.After their expressions,the cells were infected by BmNPV for another 72 h.We collected cells to detect the replication of BmNPV and found that the inhibitory effects ofBm Smt3-TCTP,Bm TCTP-WT and BmTCTP-K164 R on BmNPV were gradually weakened.These results demonstrated that BmTCTP SUMOylation could enhance the immune function of itself.UBC9,as an SUMO-conjugating enzyme,plays an important catalytic role in SUMOylation.We detected the expression of BmTCTP by up-regulating and down-regulating the expression of BmUBC9 after infection of BmNPV in Bm N cells.The results showed that the overexpression of Bm UBC9 significantly inhibit the down-regulation of BmTCTP expression.Then we examined the effect of BmUBC9 on the SUMOylation of BmTCTP during the immune response at the protein level.The result showed that BmUBC9 enhanced the SUMOylation of BmTCTP after infection of Bm NPV,and the SUMOylation of BmTCTP was increased at the early phase and then decreased.These results indicated that the SUMOylation can enhance the immune function of BmTCTP and that BmUBC9 plays an important regulatory catalytic role in this immune process.3.Identification of the relationship between TCTP and ILFIn order to understand the immune process of BmTCTP,we selected the interleukin enhancer binding factor(Bm ILF)involving in the immune process from proteins that specifically interact with BmTCTP identified by LC-MS/MS in silkworm.First,we detected the replication of BmNPV after overexpression of BmILF and infection of the Bm NPV in BmN cells.The result showed that Bm ILF was able to effectively inhibit the replication of BmNPV.At the same time,we verified that weak protein interactions occured between BmTCTP and BmILF mainly in the nucleus through Co-IP and cell colocalization experiments.Furthermore,we found that overexpression of BmTCTP up-regulated the expression of Bm ILF and that BmTCTP could significantly increase the expression of Bm ILF after infection of BmNPV.These data revealed that Bm TCTP can regulate the expression of BmILF to participate in the immune response.Taken together,we summarized that BmSmt3/BmUBC9 could participate in the SUMOylation of BmTCTP,and BmTCTP is able to regulate the expression of downstream gene BmILF to be involved in immune responses after infection of the Bm NPV in BmN cells.When BmN cells were infected with BmNPV,BmTCTP can enter the nucleus and inhibit the replication of BmNPV.Meanwhile,BmUBC9 catalyzes the SUMOylation of BmTCTP to maintain the stability of the protein in the nucleus.Furthermore,BmTCTP regulates the expression of downstream gene BmILF,and BmILF regulates other immune proteins to inhibit replication of BmNPV.