Mechanistic Study Of Protein Stability Regulated By The UFBP1,the Key Protein In Ufmylation

Background and aims:UFM1(Ubiquitin-fold modifier 1)is one of the newly identified ubiquitin-like proteins(Ubls).Similar to ubiquitin,UFM1 is conjugated to its target proteins by a three-step enzymatic reaction.Recent findings demonstrate that,UFM1 is closely related to the development of many diseases,such as breast cancer,haematological diseases,neurological diseases and cartilage development,etc.Currently,UFBP1(UFM1-binding and PCI domain-containing protein 1)and ASC1(Activating signal cointegrator 1)are the two identified targets of UFM1 UFBP1 is the first identified target of UFM1 Interestingly,studies find that in the presenbce of 17?-estradiol,the ASC1 ufmylation must be regulated by UFBP1.It is also reported that UFBP1 knockout mice result in embryo death.These results indicate that UFBP1 plays important roles in the UFM1 modification system or other biological processes.However,the functional study of UFBP1 is still very limited.Therefore,we use a label-free quantitative proteomic approach to identified UFBP1-interacting proteins and explore the mechanism of UFBP1 on the regulation of protein stability.Methods:First,the UFBP1 plasmid containing a FLAG tag at the N-terminus was constructed by standard molecular cloning techniques and transfected into HEK293T cells.Then cells were harverst to prepare cell lysates and FLAG-UFBP1 and its interacting proteins were purified with FLAG M2 affinity gel.The purified samples were separated by SDS-PAGE and visualized by silver staining.The gels were excised and proteins were digested with trypsin to obtain peptides for mass spectrometric analysis.The UFBP1-interacting proteins were obtained by label-free quantification.Bioinformatics analyses were performed to obtain the biological process of the UFBP1-interacting proteins.Biochemical methods were used to verify several interacting proteins and to investigate the specific mechanism of protein stability regulated by UFBP1.Results:Immunoprecipitation and label-free quantitative proteomics successfully identified UFBP1-interacting proteins in HEK293T cells.Three biological replicates resulted in about 81 UFBP1 interacting proteins.The bioinformatics analyses suggested that the UFBP1 interacting proteins mainly involved in the regulation of protein folding,stability,and trafficking.Biochemical experiments discovered that UFBP1 could reduce the stability of its interacting proteins through the regulation of their ubiquitination.Furthermore,using a model interacting protein,we demonstrated that UFBP1 could promote its ubiquitination by enhancing the interaction between the protein and its E3 ligase,thus down-regulating the expression level of this protein.Conclusion:We use immunopurificatioin and mass spectrometry to identify UFBP1 interacting proteins and use biochemical approaches to discover a novel mechanism by which UFBP1 regulates protein ubiquitination and degradation.UFBP1 can promote the ubiquitination of its interacting proteins by enhancing the interaction between the protein and its E3 ligase,and then reduce the expression level of the protein.This result improves our understanding of the biological function of UFBP1 and provides a basis for further exploration of the potential functions for UFBP1.