Ubiquitin-like Protein FAT10 Influences DNA Damage Repair By Modifying PCNA

Backgrounds: DNA damage may come from the intrinsic factors of the body,such as reactive oxygen species,byproducts of cellular metabolism,and mismatches due to inactivation of topoisomerase during replication in DNA;it may also come from ionizing radiation(IR),UV Light exposure(UV)and other carcinogens in nature and other external factors.DNA damage can lead to gene mutation and cell senescence.The mechanism by which DNA damage occurs and its precise and efficient repair is called the DNA damage response mechanism(DDR),which protects the body from the adverse effects of DNA damage.Proliferating cell nuclear antigen(PCNA)plays a central role in DNA damage repair mechanism.PCNA as a stable "platform" in the process of injury repair recruited a series of replication-related proteins.Ubiquitin-like proteins(UBLs),is a kind of small protein family similar to ubiquitin.It is reported that ubiquitin proteins contain homologous domains homologous to ubiquitin,with a homology of about 15% to 16%.UBLs can be divided into two subfamilies: ubiquitin domain family of UDP and ubiquitin family of modified family of proteins ?LM.UDP can be non-covalently associated with ubiquitin and ubiquitin-modified proteins.?LM has a domain homologous to the ubiquitin monomer or dimer and can be covalently bound to the substrate protein thro?gh the C-terminal glycine group under the catalysis of the E1-E2-E3 enzyme system,represented by ISG15,FUB1,NEDD8,S?MO,Urm1,UBL5,Ufm1 and FAT10.It has been reported that a variety of post-translational modifications,including ubiquitination and ubiquitination,regulate DNA damage repair by modifying PCNA when the cells in the replication experience DNA damage.When the epidermal cells are exposed to ultraviolet(UV)radiation for a long time,the RAD6-RAD18 complex can mediate a high degree of ubiquitination of the lysine residues at position 164 of PCNA,leading to changes in replicative DNA polymerase.FAT10(HLA-F locus adjacent transcript 10)is a protein with a size of 18 k D.Its N and C ends are similar to ubiquitin.The N-terminal is 29% identical to ubiquitin,while the C-terminal has 36 %the same.Encoding in human major histocompatibility complex(MHC)class I loci.Human FAT10 is expressed in mature dendritic cells,B cells,and immune organs such as thymus and spleen,but it can also be stimulated by various proinflammatory factors(IFN-?,TNF-?)in various tissues,The binding of the terminal covalently modifies the target substrate.In the preliminary work we identified by mass spectrometry that the proliferating cell nuclear antigen(PCNA)was a covalently modified substrate of FAT10.Object: To study the covalent modification mechanism of FAT10 and PCNA in DNA damage repair process,and verify whether FAT10 could covalent modify ENO1 in cells,to find a new regulation method of DNA damage repair as well as the occurance and the development of cancer,and to provide a new idea for the study of aging physiology.Methods:(1)The cells were treated with UV / IR and VP-16.The DNA damage was induced by western-blotting and q RT-PCR.The expression of FAT10 was detected by immunoprecipitation test.When the DNA was damaged,the FAT10 protein was covalently The PCNA was detected by ubiquitination degradation assay and si RNA knockdown assay to detect whether PCNA was degraded by 26 S protease by FAT10.The interaction between FAT10 and PCNA was investigated by immunofluorescence assay.The interaction between PCNA and FATNA The effect of cell formation on cell aggregation.(2)Thro?gh the construction of p Flag-CMV-eno1 and p CMV-Myc-fat10 eukaryotic expression plasmids,the two plasmids were co-transfected into HEK293 cells.Immunoprecipitation method was used to investigate whether FAT10 was covalently Modified ENO1.Rseults: First,we treated cells with UV / IR and VP-16,detected DNA damage by western-blotting and q RT-PCR.It was found that DNA damage induced the up-regulation of FAT10 expression,and the expression of FAT10 Aggravated and raised.In this experiment,when the UV radiation dose reached 20 J / m2,IR radiation dose reached 20 Gy,VP-16 dose reached 200?M,FAT10 expression of the highest.He La cells treated with UV irradiation at 10 J / m 2 and 20 J / m 2 were detected by immunoprecipitation assay,and FAT10 was found to covalently modify PCNA.Similarly,we observed that FAT10 could covalently modify PCNA when irradiated with IR and treated cells with VP-16.In the PCNA degradation assay,we found that the expression level of PCNA was gradually decreased with the increase of cytokine concentration,and the expression level of PCNA was decreased in the cells treated with 26 S proteasome inhibitor MG132.Similarly,degradation of PCNA and FAT10 in VP-16 treated cells can also be inhibited by MG132.The rseults showed that FAT10 could degrade PCNA thro?gh 26 S proteasome when the DNA was damaged by transfection of FAT10 si RNA into the cells treated with VP-16 and knocked down endogenous FAT10.Secondly,we isolated the cytoplasm and nucleus of the sample and detected the expression of PCNA and FAT10 by Western blotting.We found that most PCNAs accumulated in the nucleus.When the 26 S proteasome inhibitor was added,the accumulation of PCNA in the cytoplasm increased significantly.We also found that FAT10 was significantly accumulated in the nucleus and cytoplasm.These data indicate that FAT10 may be degraded in the cytoplasm by 26 S proteasome-mediated PCNA after treatment with VP-16.We then examined the nuclear foci by laser confocal microscopy,observed that FAT10 and PCNA were co-located at the site of injury,and that the number of nucleus aggregation sites increased when the cells were treated with VP-16,and the PCNA Expression is reduced in cytoplasm.When MG132 was added,the number of nuclear aggregates decreased and the expression of PCNA returned to normal levels.These experimental rseults indicate that FAT10 mediates PCNA degradation in the cytoplasm during DNA damage repair,which affects the increase in the number of nuclear aggregates.In addition,we constructed p Flag-CMV-ENO1 and p CMV-Myc-FAT10 expression plasmids,and these two plasmids were co-transfected into HEK293 cells.Thro?gh immunoprecipitation,it was found that FAT10 could covalently modify ENO1 in cells.Conclusion: We speculate that the DNA damage can induce the up-regulation of FAT10 and covalently modify the PCNA protein and mediate the degradation of PCNA in the cytoplasm by 26 S proteasome,thus elevating the nuclear damage site.In addition,we found that the ubiquitin-encoding protein FAT10 covalently modifies ENO1 in cells.The above rseults s?ggest that FAT10 may affect the occurrence and metastasis of tumors by covalently modifying PCNA and ENO1.