Molecular Mechanism Of DNA Damage Response Mediated By Ubiquitination Enzyme USP39

Objective The occurrence of malignant tumor is closely related to the absence of DNA damage response system.The delve study molecular mechanism of DNA damage response can facilitate the understanding of the pathogenesis of tumor and provides a reliable theoretical basis for the treatment of tumor and the development of new chemotherapeutic drugs.The DNA-damage response system,CHK2 is an important damage signal-mediate and key factor regulating repair of DNA injury.It is reported that ubiquitination and phosphorylation of CHK2 are involved in regulation of DNA damage reaction mechanism,but the study on the deubiquitination of CHK2 is not clear at present.The research of this topic focused on the role of deubiquitination modification CHK2 pathogenesis of tumor and drug resistance.Methods 1.The GeneCards database was used to screen out ubiquitinated enzymes that may interact with CHK2.After above plasmid was transfected into HEK-293 T cells for 48 hours,the cells were collected for the co-immunoprecipitation experiment,and western blot was used to detect the ubiquitination enzyme that could interact with CHK2.2.The stable interference cells line of USP39 shRNA and CHK2 shRNA were constructed by lentivirus vector shRNA.western blot was used to detect the change of CHK2 protein level after downregulation of USP39 gene.3.The plasmid of USP39 and CHK2 were respectively transfected into HEK-293 T cells,and the cells were collected for the semi-endogenous co-immunoprecipitation experiment,western blot was used to detect the interaction between CHK2 and USP39.The wild type GST-USP39-WT protein was express and purify from the coli expression strains BL21 and the HEK-293 T cell lysate transfected with the plasmid of Flag-CHK2 in vitro,and western blot was used to detect the protein interaction.4.To construct prokaryotic expressive vector of USP39 and its fragments,the full length GST-USP39-FL and its fragments GST-USP39-1(1-219aa)and GST-USP39-2(220-569aa)fusion protein were expressed and purified from the coli expression strains BL21 and the HEK-293 T cell lysate transfected with the plasmid of Flag-CHK2 in vitro,and western blot was used to detect the protein interaction region.5.The plasmid of USP39 and CHK2 were respectively transfected into HEPG2 cells,and the ubcellular localization of USP39 and CHK2 were detected by immunofluorescence assay.6.The USP39 shRNA stable cell lines were treated with the proteasome inhibitor MG132,CHX and the plasmid of USP39,and the protein levels of CHK2 and USP39 were detected by western blot.7.Enzyme catalyzed inactivation mutants(USP39 306 th amino acid loci mutants,USP39C306A)was constructed with the point mutation kit,the wild type GST-USP39-WT protein and inactivation mutants USP39C306 A protein were express and purify from the coli expression strains BL21 and the HEK-293 T cell lysate transfected with the plasmid of Flag-CHK2 in vitro,and western blot detected the polyubiquitination level of CHK2.The overexpressing plasmids of His-ub with USP39-CA and USP39-WT was respectivelyt ransfected into HEK-293 T cells,western blot was used to detect the level of ubiquitination of CHK2.8.The soft agar and flow cytometry were used to detect the cell growth,apoptosis and cell cycle arrest of lung cancer cells after downregulation USP39 and CHK2.9.To detect the expression of USP39 and CHK2 in lung cancer tissues through immunohistochemical staining experiment.Results1.The experiment of semi-endogenous immunoprecipitation showed that USP39 and CHK2 interact with each other.The GST Pull-down experiment further indicated that CHK2 and USP39 interact directly with each other.At the same time,the immunofluorescence experiment showed that USP39 and CHK2 were Co-located in the nucleus.2.The lentiviral vector shRNA mediated down regulation of USP39 significantly reduced CHK2 protein level and increased CHK2 ubiquitination level.3.Downing regulation of USP39 significantly reduced CHK2 protein level and increased CHK2 ubiquitination level by lentiviral vector shRNA.4.The deubiquitination experiments showed that the wild-type USP39 could deubiquitinate CHK2 and the enzyme catalyzed inactivation USP39 CA lost the ability to deubiquitate CHK2.5.The experiment results of soft agar and flow cytometry showed that down regulation of USP39,CHK2,and simultaneous downregulation of USP39 and CHK2 could promote the growth,inhibit cell apoptosis and weaken cell cycle arrest of lung cancer cells.6.The results of immunohistochemistry showed that CHK2 and USP39 were low expression and there was a strong correlation in lung cancer tissues.The high expression of CHK2 and USP39 could improve the survival rate of the patients in lung cancer.Conclusion 1.The USP39 can stabilizes CHK2 protein level by deubiquitination,promotes apoptosis and promote cell growth of lung cancer cell.2.The CHK2 and USP39 are low expressed in lung cancer tissues,and the high expression of CHK2 and USP39 can increase the survival rate of patients in lung cancer patients.