Proteomic Analysis And Functional Study Of UFM1 Modification System

Background and aims:Ubiquitin-fold modifier 1(UFM1) is one of the recently discovered ubiquitin-like proteins. Like ubiquitin, UFM1 forms covalent conjugates on substrates and this process is called ufmylation. The role of ufmylation is implicated in endoplasmic reticulum stress, cell homeostasis, etc. This process is linked to many diseases, such as ischemic heart disease, hip dysplasia, and cancer. In breast cancer cells, UFM1 is first transferred to a known substrate, UFBP1(UFM1-binding and PCI domain-containing protein 1) from the E3 ligase and then is further transferred to a new substrate ASC1(Activating signal cointegrator 1) in the presence of 17?-estradiol. This phenomenon indicates that the ufmylation of other proteins may be mediated by UFBP1. This not only suggests ufmylation may have two different conjugating pathways, but also implies that UFBP1 plays important roles in ufmylation. In this thesis, we first used a spectral counting based label-free quantitative proteomic approach to identify UFBP1-interacting proteins and to study the potential biological functions of UFBP1; then we explored the molecular mechanism of ufmylation; third, using biochemical and quantitative proteomic approach to identify some potential UFM1 substrates.Methods:We constructed a FLAG-UFBP1 plasmid, expressed FLAG-UFBP1 in HEK293 T cells, isolated UFBP1-interacting proteins by immunoprecipitation, digested them to obtain peptides for mass spectrometry analysis, used bioinformatic analysis and protein interaction network analysis to study the biological functions of UFBP1 and validated them by immunoblotting. We constructed a Myc-Strep-UFL1 plasmid, expressed Myc-Strep-UFL1 in HEK293 T cells, isolated UFL1- interacting proteins by affinity purification for proteomic identification, identified UFL1- interacting proteins and proteins interacting with both UFL1 and UFBP1, identified potential UFM1 substrates for the study of the molecular mechanism of ufmylation. We constructed and expressed FLAG-UFC1-UFM1 and its mutant FLAG-UFC1-UFM1 G83 X, isolated the UFM1-modified peoteins by FLAG-M2 beads, and analyzed them by liquid-chromatography coupled mass spectrometer.Results:We identified 214 UFBP1 interacting proteins by using a spectral counting based label-free quantitative proteomic approach, and found that UFBP1 interacting proteins form many protein interaction complexes. KEGG signal pathway analyses discovered that UFBP1 interacted with multiple proteasome subunits. UFBP1 could regulate the stability of its interacting proteins through three different manners: upregulation, no effect and downregulation. UFBP1 downregulated its interacting proteins may work through the ubiquitin-proteasome pathway. There were 94 UFL1-interacting proteins and 18 proteins interacted with both UFL1 and UFBP1. Use of fusion protein FLAG-UFC1-UFM1 resulted in relative high abundant UFM1 modified proteins.Conclusion:We found a novel function for UFBP1 besides its role in ufmylation. UFBP1 can regulate the stability of its interacting proteins through three different manners, and the downregulation of its interacting proteins may work through ubiquitin-proteasome pathway. UFL1-interacting proteins and proteins interacted with both UFL1 and UFBP1 were used to study the molecular mechanism of ufmylation. UFM1 fusion protein has been used to study the substrates of ufmylation. All of these would provide an important foundation for the functional study of ufmylation.