Study On Stability And Degradation Mechanism Of C1orf109 Protein 265/280 Amino Acid Transcript

In mammalian cells,the rate of protein renewal must be strictly controlled,because many newly synthesized proteins will degrade rapidly,and damaged or misfolded proteins also need to be rapidly degraded to maintain cell metabolism.The ubiquitin-proteasome system is a specialized protein hydrolysis system that controls protein degradation and plays an important role in maintaining protein homeostasis in the cell.It is important not only in regulating protein homeostasis,but also in handling many cell regulators related to DNA damage and repair,cell proliferation and survival,cell differentiation,and drug resistance.As a new protein molecule,C1orf109,its biological functions and potential molecular regulatory mechanisms are still unclear.In previous studies,it was found that C1orf109 protein was low in various tumor cells,but the reason for its low expression in tumor cells is not yet clear.Therefore,this subject conducted a preliminary study on the stability of the 265 amino acid transcripts(C1orf109-265)and 280 amino acid transcripts(C1orf109-280)of the C1orf109 protein and the molecular regulation mechanisms of their degradation.In this paper,cycloheximide was used to treat He La and HEK-293 T cells to inhibit protein synthesis and detect the half-life of C1orf109-265 and C1orf109-280 proteins.It was found that the half-life of C1orf109-265 protein was longer than that of C1orf109-280.C1orf109-265 protein is relatively stable in both cells.In order to explore the degradation pathway of C1orf109 protein in cells,He La and HEK-293 T cells were treated with proteasome inhibitor MG132,and it was found that after 4 hours of treatment with MG132,C1orf109-265 and C1orf109-280 proteins both accumulated significantly,indicating that the two both can be degraded by the proteasome pathway.Next,using ubiquitin molecules and C1orf109-265 and C1orf109-280 proteins under complete denaturation,respectively,co-immunoprecipitation experiments were carried out to detect the ubiquitination status of the two,and it was found that both have ubiquitination modification.In order to explore the molecular regulation mechanism of ubiquitination modification of C1orf109 protein,the most important thing is to find E3 ligase that mediates its ubiquitination.Screened in the mass spectrometry results of C1orf109-265 and C1orf109-280 proteins,three protein molecules with E3 ligase function were obtained,namely TRIM21,UBR5 and STUB1.Next,the interaction between the three E3 ligases and C1orf109 protein was verified by immunoprecipitation technology,and it was found that STUB1 interacted with C1orf109-280.In this study,a recombinant STUB1 was constructed,and exogenous STUB1 was introduced into the cell to detect the change in the expression of the substrate protein C1orf109-280 protein.It was found that overexpression of STUB1 can downregulate the expression level of C1orf109-280 protein.Then the cytoplasmic separation technology was used to study the cell localization of C1orf109-280 degradation,and it was found that C1orf109-280 protein mainly exists in the nucleus,and STUB1 promotes its degradation in the nucleus.After we silenced STUB1 with si RNA,the expression level of C1orf109-280 protein reverted,further indicating that C1orf109-280 is the substrate protein of STUB1.In summary,this study conducted a preliminary study on the stability and degradation mechanism of C1orf109-265 and C1orf109-280.It was found that in He La and HEK-293 T cells,the half-life of C1orf109-265 protein was longer than that of C1orf109-280 protein.The former is relatively stable.The study found that both C1orf109-265 and C1orf109-280 proteins were degraded by the ubiquitin-proteasome pathway,and found that STUB1 could mediate the ubiquitination modification of C1orf109-280 protein.The degradation mechanism of C1orf109-265 and C1orf109-280 proteins was preliminarily clarified.It laid the foundation for the follow-up study of the biological function of C1orf109 protein.