The Study On ZNRF3 Ubiquitin-Mediated Protein Degradation And Its Relationship With Cancer

ZNRF3(zinc and ring finger 3)is a transmembrane protein containing a RING domain and function as a transmembrane E3 ubiquitin ligase which regulate two important membrane receptors FZD and LRP6 in the Wnt/β-catenin pathway.The mechanism of how it inhibited the Wnt signaling pathway has been revealed.However,it is still unclear which molecules exist in the upstream of ZNRF3 that regulate its expression level and the downstream targets of Wnt/β-catenin pathway.ZNRF3 acted as a tumor suppressor gene has been verified in multiple tumors,but its biological functions in colorectal cancer which closely related to the Wnt signaling pathway is still unclear.To reveal the ZNRF3 biological functions and its protein expression regulation mechanisms.Structure and function analysis indicating the ZNRF3 was a very important.Detection of protein expression among a variety of colorectal cancer cells indicated that low expression of ZNRF3 in DLD1 and U2 OS cells,whereas ZNRF3 was highly expressed in HCT116 and HeLa cells.Therefore,ZNRF3 overexpression were conducted in DLD1 and U2 OS cells to detect ZNRF3 proliferation,colony formation and migration ability.Results confirmed that ZNRF3 could inhibited the proliferation and migration of tumor cells.Meanwhile,knockdown experiments were performed to detect the above phenotype and found they were consistent with the results of overexpression ones.Furthermore,we used Western-blot to detect that overexpression of ZNRF3 could lead to inhibition of cell cycle progression and epithelial-mesenchymal transition meanwhile promote apoptosis.To identify the precise degradation mechanism of ZNRF3,cells were treated with the proteasome inhibitor MG132 and the Cullin family protein inhibitor MLN4924,confirming that ZNRF3 was degraded by the ubiquitin-proteasome pathway and that its protein level was regulated by the SCF E3 ligase complex.Furthermore,Co-IP method were used to confirm ZNRF3 could bind to the Cullin1,Rbx1,and Skp1 proteins.And under the condition of knockdown of Cullin1,ZNRF3 expression could be increased and its half-life was extended.It was clarified that Cullin1 could regulate ZNRF3 protein expression level.Then His Pull-down experiments verified that the ubiquitination linkage types of ZNRF3 were K27 and K63.Since the F-box protein played a key role in binding to the substrate,Co-IP analysis indicated that β-TRCP could specifically bind to ZNRF3.Moreover,the degradation assay confirmed that ZNRF3 could be degraded by β-TRCP1,and this process could be rescued.After knockdown of β-TRCP1,the expression and half-life of ZNRF3 were significantly increased,and β-TRCP1 could increase the ubiquitination level of ZNRF3.Above experiment results revealed that β-TRCP1 which acted as an F-box protein in the SCF complex can directly bind to ZNRF3 on its upstream and was the key molecule regulating the ubiquitination and degradation of ZNRF3.Meanwhile,the downstream signal of Wnt/β-catenin was inhibited by knocking down β-TRCP or adding CKI inhibitors,which further identified that ZNRF3 was the substrate of β-TRCP.As a component of the SCF E3 ubiquitin ligase complex that binds directly to substrate proteins,β-TRCP1 generally tended to recognize a conserved degron sequence which was D-pS-G-X-X-pS.Meanwhile,serine in this sequence would be further phosphorylated so that β-TRCP1 could recognizes it.Therefore,using dephosphorylation treatment experiments,kinase degradation assay,and knockdown kinase experiments was confirmed the upstream molecule which controlled the phosphorylation of ZNRF3 was CKIδ.Using a combination of multiple sequence alignment and degron mutation assay,it was revealed that the degron of ZNRF3 was SSGQCHCS.In addition,it was found that ZNRF3 negatively regulates the Wnt/β-catenin signaling pathway upon degron mutation.In summary,our results demonstrated the biological functions of ZNRF3 as a tumor suppressor in colorectal cancer.And also its ubiquitination and degradation mechanism.It was further confirmed that the Skp1-Cullin1-β-TRCP1 protein complex binds to and degradated ZNRF3.Furthermore,the SCF E3 complex could recognize the degron sequence in a phosphorylation-dependent manner.Moreover,improve our understanding of the Wnt pathway,under Wnt off conditions,β-TRCP formed a degradation complex to recognize and degrade CKIα/GSK3-mediated phosphorylated form of β-catenin,to repress β-catenin targeted genes,and maintain Wnt in silent status.Under Wnt on conditions,β-TRCP1 could promote the degradation of CKI-mediated phosphorylated form of ZNRF3,to sustain the Wnt pathway.