Expression Of TRIM50 In Ovarian Cancer Tissues And Its Effect In Vitro And In Vivo And Its Molecular Mechanism

BackgroundOvarian cancer is the gynecological malignant tumor with the highest mortality rate,of which epithelial ovarian cancer is the most important pathological type.The onset of epithelial ovarian cancer is insidious and it progresses rapidly.Most patients are in advanced stage at the time of initial diagnosis.Surgical treatment is only effective for patients in early stage,and most patients are not sensitive to conventional chemotherapy and radiotherapy.Therefore,it is of great significance to find new interventions for ovarian cancer.The ubiquitin proteasome system(UPS)is an important pathway for the selective degradation of proteins in eukaryotic cells.UPS consists of ubiquitin(Ub),26S proteasome,E1 ubiquitin activating enzyme,E2 ubiquitin-binding enzyme,E3 ubiquitin ligase and DUBs ubiquitin dissociation enzyme.Among them,E3 ubiquitin ligase determines the specific selection of substrate and is the key enzyme of UPS.Currently,the development of drugs based on the composition of UPS is a hot spot in cancer research.TRIM(tripartite motif-containing protein)family is a class of RING-type E3 ubiquitin ligase,which is characterized by a N-terminal RING-finger,one or two B-box domains,and a coiled-coil domain.TRIM proteins have complex C-terminal structures.In recent years,TRIM family has received increasing attention due to its unique structure and powerful biological functions.Some TRIM proteins are involved in the occurrence and progression of tumors by targeting tumor-related molecules.TRIM50 is a new member of the TRIM family.It has been reported that TRIM50 is involved in the regulation of nervous system development,gastric acid secretion and autophagy.There is few report on the function of TRIM50 in tumors.Only one report said that TRIM50 inhibited epithelial-mesenchymal transition of hepatocellular carcinoma by degrading Snail.The function of TRIM50 in ovarian cancer has not been reported so far.Src protein is a member of the Src family kinases(SFKs)and is a non-receptor protein with tyrosine kinase activity encoded by a proto-oncogene.The structure of Src kinase consists of a single sequence(U),an SH3 domain,an SH2 domain,a junction segment,an SH1 domain(protein tyrosine kinase domain)and a carboxy-terminal regulatory tail.Activation of Src is regulated by dephosphorylation of the Tyr530 in carboxy-terminal regulatory tail and autophosphorylation of the Tyr419 in kinase domain.Src can be activated by a variety of receptor tyrosine kinases and integrins,participate in the cell signaling cascades and play a key regulatory role in maintaining cell homeostasis.Studies have shown that Src kinase is overexpressed or highly activated in a variety of tumors such as prostate cancer,breast cancer and ovarian cancer.Abnormally activated Src kinase over-activates signaling pathways such as mitogen-activated protein kinase(MAPK),phosphatidylinositol 3-kinase(PI3K)and protein kinase B(Akt/PKB)and signal transducer and activator of transcription 3(STAT3),and promotes proliferation,resistance to apoptosis,angiogenesis,invasion and metastasis of tumors.Therefore,Src is a promising target for anti-tumor intervention,and regulation of Src may provide a new strategy for reversing the progression of ovarian cancer.In this study,the expression of TRIM50 protein in ovarian cancer tissue samples was detected and its clinical significance was analyzed.The effect of TRIM50 on the malignant behaviors of ovarian cancer cell lines and the growth of xenograft tumors in nude mice were investigated.The molecular mechanism involved in the targeting and regulating of Src protein by TRIM50 was further explored.Part I Expression of TRIM50 in ovarian cancer tissues and its effect in vitro and in vivoObjective:1.To detect the expression of TRIM50 protein in ovarian cancer tissues and analyze its clinical significance.2.To study the effect of TRIM50 on the proliferation,colony formation and migration of ovarian cancer cells,and its effect on the growth of xenograft tumors in nude mice.Methods:1.To investigate the expression of TRIM50 protein in ovarian cancer tissue specimens and its clinical significanceThe expression of TRIM50 protein in serous adenocarcinoma tissues of 67 patients were detected by immunohistochemical staining(IHC).The correlation of TRIM50 level with clinical stage and pathological grade was statistically analyzed.2.To investigate the effect of TRIM50 on the malignant behaviors of ovarian cancer cells2.1 Construction of the loss-of-function and gain-of-function ovarian cancer cellular modelsFor the construction of loss-of-function ovarian cancer cellular model,TRIM50-specific Si-RNA(Si-TRIM50)was transiently transfected into A2780 and SK-OV-3 ovarian cancer cell lines.The cells transfected with unintentional sequence(Si-NC)and untransfected cells(Blank)were used as controls.For the construction of gain-of-function ovarian cancer cellular model,TRIM50 plasmid was transiently transfected into the A2780 and SK-OV-3 cell lines.The cells transfected with the empty vector and untransfected cells(Blank)were used as controls.The blocking efficiency of Si-TRIM50 and the overexpression efficiency of TRIM50 plasmid were verified by western blot.2.2 To investigate the effect of TRIM50 on proliferation,colony formation and migration capabilities of ovarian cancer cellsThe CCK-8 assay,colony formation assay and transwell assay were used to detect the effect of TRIM50 on proliferation,colony formation and migration capabilities of ovarian cancer cells.3.To investigate the effect of TRIM50 on the growth of ovarian cancer xenograft tumors in nude mice3.1 Construction of ovarian cancer xenograft tumor models in nude miceOvarian cancer cells were inoculated into both flanks of the female BALB/c nude mice(4-6 weeks).SK-OV-3 and A2780 cells were used to construct xenograft tumor models derived from two human ovarian cancer cell lines.3.2 To investigate the effect of TRIM50 on the growth of ovarian cancer xenograft tumorsAfter visible tumors appeared,tumors in the right flank were injected with 30 ?g of TRIM50 plasmid every other day,and tumors in the left flank were injected with empty vector(30?g)as mock control group.Tumor volumes were recorded periodically and tumor growth curves were plotted.After the nude mice were sacrificed,the volume and weight of formed tumors on both flanks were analyzed.Result:1.The expression of TRIM50 protein in ovarian cancer tissue specimens and its clinical significanceIHC was used to detect the expression of TRIM50 protein in clinical ovarian serous adenocarcinoma tissues of 67 patients.The results showed that TRIM50 was primarily localized in the cytoplasm of ovarian cancer cells.Statistical analysis data showed that TRIM50 level in ovarian cancer tissues of patients in FIGO stage II,III and IV was significantly lower than that of patients in FIGO stage I.Compared with ovarian cancer tissues in G1(highly differentiated),TRIM50 expression was significantly decreased in ovarian cancer tissues in G2(moderately differentiated)and G3(poorly differentiated).Correlation analysis data showed that TRIM50 level in ovarian cancer tissues was significantly inversly correlated with clinical stage and pathological grade.These results suggested that loss of TRIM50 expression contributed to the disease progression of ovarian cancer.2.Effect of TRIM50 on proliferation,colony formation and migration of ovarian cancer cellsThe loss-of-function and gain-of-function ovarian cancer cellular models were constructed.CCK-8 assay,colony formation assay and transwell assay were used to detect the malignant behaviors of ovarian cancer cells.The results showed that the proliferation,colony formation and migration capabilities of ovarian cancer cells were significantly up-regulated after knockdown of TRIM50 expression.After overexpression of TRIM50,these malignant behaviors of ovarian cancer cells were significantly inhibited,which indicated that TRIM50 exerted as a tumor suppressor in ovarian cancer cells.3.Effect of TRIM50 on the growth of ovarian cancer xenograft tumors in nude miceThe ovarian cancer xenograft tumor models in nude mice were constructed.Tumors in the right flank of nude mice were injected with TRIM50 plasmid every other day,and tumors in the left flank were injected with the same amount of empty vector as control group.The tumor growth curve showed that the growth of ovarian cancer tumors was significantly inhibited after injection of the TRIM50 plasmid compared to the control group.After the tumors were isolated,it was found that the weights and volumes of the tumors injected with TRIM50 plasmid were significantly smaller than that of the control group,confirming the tumor suppressor role of TRIM50 in vivo.Conclusion:1.The expresson level of TRIM50 protein in ovarian cancer tissue samples was inversely correlated with the clinical stage and pathological grade of ovarian cancer.2.TRIM50 significantly inhibited the proliferation,colony formation and migration capabilities of ovarian cancer cells,and significantly inhibited the growth of ovarian cancer xenograft tumors in nude mice.Part II The binding and regulatory effects of TRIM50 on the Src proteinObjective:1.To screen the target of TRIM50,and investigate the direct binding of TRIM50 to Src prorein and the domains that mediated their interaction.2.To investigate the regulatory effect of TRIM50 on Src protein in ovarian cancer,and the mechanism by which TRIM50 exerted its tumor suppressor effect by regulating Src.Methods:1.To investigate the direct binding of TRIM50 to Src protein1.1 Immunoprecipitation experiment(IP)IP assay was used to screen for tumor-related molecules that might interact with TRIM50.It was found that TRIM50 bound to the proto-oncogene Src protein.1.2 Colocalization experimentTRIM50 and Src plasmids were co-transfected into ovarian cancer cells for immunofluorescence experiment.The colocalization of TRIM50 and Src protein was detected by laser confocal microscopy.1.3 In vitro translation experimentTRIM50 and Src proteins were synthesized via in vitro translation experiment,and the direct interaction between the synthesized TRIM50 and Src proteins was detected by IP assay.2.To investigate the domains that responsible for the interaction between TRIM50 and Src proteins.2.1 To investigate the domain of TRIM50 protein responsible for the interaction between TRIM50 and SrcA series of domain-deleted mutants of TRIM50 were constructed and each TRIM50 mutant was co-transfected with Src plasmid into HEK293T cells,respectively.The cells transfected with Src and wild-type TRIM50 plasmid were used as the positive control.IP assay was performed to detect the binding of each TRIM50 mutant to Src,in order to explore the domain of TRIM50 mediating the interaction between TRIM50 and Src proteins.2.2 To investigate the domain of Src protein responsible for the interaction between TRIM50 and SrcA series of domain-deleted mutants of Src were constructed.Each Src mutant was co-transfected with TRIM50 plasmid into HEK293T cells,respectively.The cells transfected with TRIM50 and wild-type Src plasmid were used as the positive control.IP assay was used to investigate the binding of each Src mutant to TRIM50 to detect the domain of Src mediating the interaction between TRIM50 and Src.3.To study the regulatory effect of TRIM50 on Src protein in ovarian cancer3.1 Western blot experimentTRIM50 was knocked down and overexpressed in ovarian cancer cells,respectively.The protein levels of TRIM50,Src and its activated form p-Src(Tyr419)were detected by western blot.The regulation of Src level by TRIM50 was detected.3.2 Cycloheximide(CHX)experimentOvarian cancer cells were transfected with TRIM50 plasmid or mock control,and were cultured for 24 h before further treatment with CHX for 0 h,2 h,4 h,6 h and 8 h.The protein levels of Src at different time points were detected by western blot.The trend of Src degradation after blocking protein synthesis was analyzed,and the effect of TRIM50 on degradation of Src was examined.3.3 To verify the regulatory effect of TRIM50 on Src in ovarian cancer xenograft tumorsThe protein of xenograft tumors in nude mice was extracted,and the expression levels of TRIM50,Src and p-Src(Tyr419)in tumors were detected by western blot,in order to verifiy the overexpression efficiency of TRIM50 in tumors and the regulatory effect of TRIM50 in vivo.3.4 To verify the regulatory effect of TRIM50 on Src by detecting ovarian cancer tissue specimens The protein levels of Src and TRIM50 in ovarian serous adenocarcinoma tissues were detected by IHC.The correlation between Src and TRIM50 protein levels was statistically analyzed,in order to verify the regulatory effect of TRIM50 on Src in ovarian cancer tissue specimens.4.To investigate the pathway of TRIM50-mediated Src degradation in ovarian cancer cellsThe intracellular pathways for protein degradation mainly include the lysosomal pathway and the ubiquitin proteasome pathway.To explore the way TRIM50 mediated the degradation of Src,ovarian cancer cells were transfected with TRIM50 plasmid or mock control and further treated with MG132 or chloroquine to inhibit proteasome and lysosomal activity respectivelly.Western blot was used to detect the level of Src expression.5.To clarity the mechanism by which TRIM50 exerted its tumor suppressor effect by regulating SrcTo further define whether TRIM50 exerted its tumor suppressor effect via negative regulation of Src,we performed a rescue experiment.We co-transfected TRIM50 and Src plasmids into ovarian cancer cells.Western blot was used to verify the overexpression of TRIM50 and Src,and transwell and colony formation assays were performed to detect migration and colony formation capabilities of these transfected cells.We investigated whether exogenous overexpression of Src could reverse the anti-cancer effect of TRIM50.Result:1.The direct binding of TRIM50 to Src protein1.1 The binding of TRIM50 to Src proteinTo explore the target of TRIM50,we used IP assay to screen for tumor-related molecules that might interact with TRIM50.It was found that TRIM50 interacted with the proto-oncogene Src in HEK293T and ovarian cancer cells,and TRIM50 interacted with both exogenous and endogenous Src proteins,indicating that Src might be the target of TRIM50.1.2 Colocalization of TRIM50 and Src proteins in ovarian cancer cellsTRIM50 and Src plasmids were co-transfected into ovarian cancer cells.The results of immunofluorescence assay showed that TRIM50 and Src proteins colocalized in ovarian cancer cells,which further verified the interaction between TRIM50 and Src.1.3 The direct interaction between TRIM50 and Src proteinsTRIM50 and Src proteins were synthesized in the in vitro translation system.The results of IP assay showed that the synthesized TRIM50 could directly bind to Src protein in vitro.2.The domains responsible for the interaction between TRIM50 and Src proteins2.1 The domain of TRIM50 protein responsible for the interaction between TRIM50 and SrcA series of domain-deleted mutants of TRIM50 were constructed and were co-transfected with the Src plasmid into HEK293T cells.IP assay showed that the B-box2 domain-deleted TRIM50 mutant failed to interact with Src,indicating that the B-box2 domain of TRIM50 was required for the interaction between TRIM50 and Src.2.2 The domain of Src protein responsible for the interaction between TRIM50 and SrcA series of domain-deleted mutants of Src were constructed and were co-transfected with the TRIM50 plasmid into HEK293T cells.IP assay showed that the SH3 domain-deleted Src mutant failed to bind to TRIM50,indicating that SH3 domain of Src was required for the binding between TRIM50 and Src.3.Regulatory effect of TRIM50 on Src protein in ovarian cancer3.1 Regulatory effect of TRIM50 on Src protein levelBased on the interaction between TRIM50 and Src proteins,we hypothesized that TRIM50 might exert its tumor suppressor effect by regulating Src protein level.TRIM50 was knocked down and overexpressed in ovarian cancer cells,respectively.Western blot results showed that knockdown of TRIM50 significantly increased the levels of Src and p-Src(Tyr419),whereas overexpression of TRIM50 reduced the abundance of Src and p-Src(Tyr419),suggesting that TRIM50 negatively regulated Src protein level and inhibited Src activity.3.2 Regulatory effect of TRIM50 on the stability of Src proteinTRIM50 plasmid or empty vector was transfected into ovarian cancer cells,and cycloheximide was used to block protein synthesis.Western blot was used to detect the degradation trend of Src protein.The results showed that TRIM50 significantly reduced the protein stability of Src in these CHX-treated ovarian cancer cells,indicating that TRIM50 promoted the degradation of Src protein.3.3 The negative regulatory effect of TRIM50 on Src in ovarian cancer xenograft tumorsThe protein levels of TRIM50,Src and p-Src(Tyr419)in ovarian cancer xenograft tumors were detected by western blot.The results showed that TRIM50 was successfully overexpressed in tumors injected with TRIM50 plasmid,and the protein levels of Src and p-Src(Tyr419)were significantly suppressed in TRIM50 overexpressed tumors,which farther demonstrated the negative regulatory effect of TRIM50 on Src in vivo.3.4 Correlation between Src and TRIM50 protein levels in ovarian cancer tissue samplesThe expression of Src and TRIM50 proteins in ovarian serous adenocarcinoma tissue samples was detected by IHC assay.The results showed that Src expression was significantly inversely correlated with the level of TRIM50 protein,which further demonstrated the negative regulatory effect of TRIM50 on Src in clinical specimens.4.Pathway of TRIM50-mediated Src protein degradation in ovarian cancer cells Ovarian cancer cells were transfected with TRIM50 plasmid or mock control and further treated with MG 132 or chloroquine to inhibit proteasome and lysosomal activity respectivelly.Our data showed that treatment of cells with MG 132 significantly rescued the negative regulation of Src by TRIM50,whereas treatment with chloroquine had no influence on the effect of TRIM50,which indicated that TRIM50 negatively regulated Src by proteasome mediated degradation.5.TRIM50 exerted anti-cancer effect in ovarian cancer by negatively regulating Src proteinThe tumor suppressor effect of TRIM50 and its negative regulation of Src were confirmed in previous studies,thus we intended to investigate whether TRIM50 exerted its anti-tumor effect through negative regulation of Src.TRIM50 and Src plasmids were co-transfected into ovarian cancer cells.Western blot assay verified successful overexpression of TRIM50 and Src in these transfected cells.We detected the colony formation and migration of these transfected cells,and our data showed that exogenous overexpression of Src significantly reversed the suppression of malignant behaviors of ovarian cancer cells by TRIM50.Thus these data confirmed that TRIM50 acted as a tumor suppressor in ovarian cancer cells via its negative regulation of Src.Conclusion:1.TRIM50 directly bound to Src protein,and the B-box2 domain of TRIM50 and the SH3 domain of Src mediated their interaction.2.TRIM50 induced the degradation of Src protein through the proteasome pathway and exerted its tumor suppressor effect by degrading Src.Part ? The molecular mechanism of TRIM50 regulating Src in ovarian cancer cellsObjective:1.To investigate the ubiquitous modification of Src protein mediated by TRIM50.2.To investigate the role of the RING domain in the ubiquitous degradation of Src and suppression of ovarian cancer by TRIM50.Methods:1.To investigate the ubiquitous modification of Src protein mediated by TRIM501.1 Ubiquitination analysis experimentThe HA-tagged Ub plasmid(HA-Ub)and TRIM50 plasmid were co-transfected into ovarian cancer cells.The ubiquitous modification of Src mediated by TRIM50 was detected by IP assay.1.2 In vitro ubiquitination experimentThe TRIM50 and Src proteins were synthesized by in vitro translation system,and the synthesized TRIM50 and Src proteins were incubated with Ub,UBE1,UbcH5a(UBE2D1),ATP and MgCl2 to form an in vitro ubiquitination system.Western blot was used to detect the ubiquitination status of Src and the E3 ubiquitin ligase function of TRIM50 was investigated.2.To investigate the type of TRIM50-mediated ubiquitous modification of Src in ovarian cancer cellsHA-UB-K48 and HA-UB-K63 plasmids were ubiquitin mutant vectors,which contain arginine substitutions of all of their lysine residues except the one at position 48 and 63,respectively.TRIM50 plasmid was co-transfected with HA-Ub-K48 or HA-Ub-K63 plasmids into HEK293T and SK-OV-3 cells.The type of TRIM50-mediated ubiquitous modification of Src was detected by IP assay.3.To investigate the role of RING domain in the TRIM50-mediated ubiquitous degradation of Src and suppression of ovarian cancer3.1 The role of RING domain in the TRIM50-mediated ubiquitination of SrcHA-Ub and RING domain-deleted TRIM50 mutant were co-transfected into ovarian cancer cells.The cells transfected with HA-Ub and wild-type TRIM50 plasmids were used as the positive control,and the cells transfected with HA-Ub and empty vector were used as the negative control.The ubiquitination status of Src was detected by IP assay,in order to investigate the role of RING domain in the ubiquitination of Src mediated by TRIM50.3.2 The role of RING domain in the TRIM50-mediated negative regulation of SrcThe RING domain-deleted TRIM50 mutant was transfected into ovarian cancer cells.The cells transfected with wild-type TRIM50 plasmid were used as the positive controls,and the cells transfected with empty vector were used as the negative control.Western blot was performed to detect the levels of Src and p-Src(Tyr419),in order to investigate the role of RING domain in the negative regulation of Src by TRIM50.3.3 The role of RING domain in the TRIM50-mediated suppression of ovarian cancer progressonThe RING domain-deleted TRIM50 mutant,wild-type TRIM50 plasmid and empty vector were transfected into ovarian cancer cells,respectively.Transwell and colony formation assays were performed to detect the migration and colony formation capabilities of the transfected cells,in order to investigate the role of RING domain in suppression of ovarian cancer by TRIM50.Result:1.The study on TRIM50-mediated ubiquitination of Src protein in ovarian cancer cells1.1 The ubiquitous modification of Src protein mediated by TRIM50HA-Ub and TRIM50 plasmids were co-transfected into ovarian cancer cells.IP assay was performed to detect the ubiquitination status of Src.The results showed that TRIM50 could tag ubiquitin chains to Src protein.1.2 The E3 ubiquitin ligase activity of TRIM50The TRIM50 and Src proteins synthesized in the in vitro translation system were incubated with Ub,UBE1,UbcH5a(UBE2D1),ATP and MgCl2 to form an in vitro ubiquitination system.The ubiquitination status of Src was detected by western blot.The results showed that TRIM50 was able to tag the ubiquitin chains to the Src protein in a simple in vitro system,indicating that TRIM50 was an E3 ubiquitin ligase that directly mediated the ubiquitous modification Src.2.The type of ubiquitination of Src mediated by TRIM50K48 and K63-linked poly-ubiquitinations are the most clarified ubiquitination types.TRIM50 plasmid was co-transfected with HA-Ub-K48 or HA-Ub-K63 plasmid into HAK293T and SK-OV-3 cells.The ubiquitination status of Src was detected by IP assay.The results showed TRIM50 could put the K48-linked poly-ubiquitin chains to Src,further confirming that TRIM50 mediated Src degradation via the ubiquitin proteasome pathway.3.The role of RING domain in the TRIM50-mediated ubiquitous degradation of Src and suppression of ovarian cancer3.1 The role of RING domain in the TRIM50-mediated ubiquitination of Src proteinHA-Ub and RING domain-deleted TRIM50 mutant were co-transfected into ovarian cancer cells.The cells transfected with HA-Ub and wild-type TRIM50 plasmids were used as the positive control,and the cells transfected with HA-Ub and empty vector were used as the negative control.IP assay was used to detect the ubiquitination status of Src.The results showed that TRIM50 could not tag ubiquitin chains to Src protein after deletion of RING domain,indicating that TRIM50 mediated Src ubiquitination via its RING domain.3.2 The role of RING domain in the TRIM50-mediated negative regulation of SrcThe RING domain-deleted TRIM50 mutant was introduced into ovarian cancer cells.The cells transfected with wild-type TRIM50 plasmid were used as the positive controls,and the cells transfected with empty vector were used as the negative control.Western blot was performed to detect Src and p-Src(Tyr419)protein levels.The results showed that the RING domain-deleted TRIM50 mutant lost the function of down-regulating Src and p-Src(Tyr419),indicating that TRIM50 relied on its RING domain to negatively regulate Src.3.3 The role of RING domain in the TRIM50-mediated suppression of ovarian cancerThe RING domain-deleted TRIM50 mutant,wild-type TRIM50 plasmid and empty vector were transfected into ovarian cancer cells,respectively.The migration and colony formation capabilities of ovarian cancer cells were detected by transwell and colony formation assays.The results showed that after deletion of the RING domain,TRIM50 lost its function of suppressing the malignant behaviors of ovarian cancer cells,indicating that TRIM50 relied on RING domain to exert its anti-tumor effect.Conclusion:1.TRIM50 induced K48-linked poly-ubiquitous modification of Src via its E3 ubiquitin ligase activity,and induced Src degradation through ubiquitin proteasome pathway in ovarian cancer.2.TRIM50 relied on its RING domain to mediate the ubiquitous degradation of Src and exert its tumor suppressor function in ovarian cancer.Innovations and limitationsInnovations:1.In this study,we investigated the function of TRIM50 in ovarian cancer and analyzed the correlation of TRIM50 expression with clinical stage and pathological grade,which provided a basis for exploring new therapeutic targets and prognostic indicators for ovarian cancer.2.We revealed that TRIM50 suppressed Src through ubiquitin proteasome pathway,which laid a foundation for clarifying the regulatory mechanism of Src and developing novel Src inhibitors.3.We explored the key domains of TRIM50 protein responsible for its suppression of Src and anti-tumor function,which provided a theoretical basis for clarifying the structural and functional characteristics of TRIM50 and designing small molecule compounds based on TRIM50 domains.Limitations:1.The number of ovarian cancer tissue specimens included in this study waits to be further expanded,so that the specimens in different stages can be distributed more evenly.Tissue specimens originated earlier,so G1,G2 and G3 pathological grading methods were used.2.The function of other domains of TRIM50 protein(Coiled-coil and PRY-SPRY domains)remains to be investigated.