| Background and aims:UFBP1(UFM1-binding and PCI domain-containing protein 1),a substrate of ubiquitin-fold modifier 1(UFM1),is composed of 314 amino acids,located on chromosome 20p13 and has a molecular weight of about 35.6 kDa.UFBP1 is a highly conserved protein that plays an important role in multicellular organisms.UFM1 modification system is a novel ubiquitin-like modification system,which covalently modifies lysine residues in target proteins.In recent years,studies have found that UFM1 modification system not only plays important roles in cell homeostasis and endoplasmic reticulum stress,but also is closely associated with many diseases,including breast cancer,blood diseases,nervous system diseases,and chondrodysplasia.A few UFM1 modified substrates have been discovered,such as UFBP1,activated signal cointegrator factor 1(ASC1),60S ribosomal protein L26(RPL26),and histone 4.UFM1-specific ligase 1(UFL1),a ligase for UFM1 modification,promotes the UFM1 modification of UFBP1 at the highly conserved residue K267.In the presence of 17β-estradiolum,UFBP1 can promote the UMF1 modification on ASC1 and this modification promotes the development of breast cancer.The UFBP1 N-terminus contains a signal peptide,which resides UFBP1 on the endoplasmic reticulum.UFBP1 also contains a PCI domain at the C-terminus,which is often found in the subunits of the proteasome,COP9 signal body,and translation initiation factor.This domain is the site for protein interaction.However,it is unclear whether mutation of UFBP1 on this domain affects its stability and whether mutation affects the proliferation,migration,and invasion of gastric cancer cells.This work aims to explore these questions.Methods:First,FLAG-tagged UFBP1 mutant plasmids(UFBP1-K267R、UFBP1-ΔPCI、UFBP1-1-273、UFBP1-1-228)were constructed and transfected into HEK293T cells as well as FLAG-UFBP1.Immunoprecipitation and immunoblotting assays were performed to investigate the effects of UFBP1 mutants on its ubiquitination and degradation through the ubiquitin-proteasome system.Then,the effects of UFBP1 and its mutants on the UFBP1-interacting proteins were determined by Western blotting analysis.The effect of UFBP1 on cell cycle was detected by flow cytometry.CCK-8 proliferation experiment was conducted to study the effect of UFBP1 and its mutants on the proliferation of a gastric cancer cell line.Would healing and transwell assays were used to investigate the migration and invasion of gastric cancer cells.The expression and effect of UFBP1 mRNA level on overall patient survival were analyzed using publically available databases.Results:Wild-type UFBP1 was more readily ubiquitinated and degraded than K267R andΔPCI mutants.MG 132 treatment increased the UFBP1 protein level but did not significantly affect the K267R and △PCI protein level.UFBP1 downregulated the protein levels of interacting proteins SF3B1 and ANT3.UFBP1 upregulated the protein levels of interacting protein EF2.The ΔPCI mutant downregulated the SF3B1 protein level.Other mutants had no regulatory effect on the protein level of SF3B1 and ANT3.K267R andΔPCI mutants but not the wild-type UFBP1 increased the SOX9 protein level.Cell cycle experiments revealed that UFBP1 could reduce the number of cells in G2/M phase.Tumor biology experiments found that UFBP1 can promote the proliferation,migration,and invasion of gastric cancer cells.Database analysis found that higher UFBP1 expression significantly reduced the overall survival rate of gastric cancer patients.Conclusion:UFBP1 is degraded by the ubiquitin-proteasome system,and lysine 267(K267)and the PCI domain are most likely the key residue and domain responseible for its ubiquitination.UFBP1 downregulates ANT3 through the PCI domain.UFBP1 does not affect SOX9 protein level but the K267R and ΔPCI mutants upregulate SOX9.UFBP1 but not the K267R mutant upregulates EF2 protein level and enhances the cell cycle progression.UFBP1 promotes the proliferation,migration,and invasion of cancer cells. |
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