HUWE1 Mediates The Neddylation Of DNA-PKcs To Promote The Repair Of DNA Double-strand Breaks

Objective:DNA-dependent protein kinase catalytic subunit?DNA-PKcs?is the core component of DNA-PK complex,which plays important role in the non-homologous end-joining?NHEJ?repair of DNA double strand breaks.The activity of DNA-PKcs is strictly controlled by its phosphorylation of post-translation modification.The ubiquitin-like protein,NEDD8 has been reported to implicate in regulation of DNA damage response.However,it remains mysterious whether and how the NEDD8-related neddylation affects DNA-PKcs and the NHEJ process.This study aims to investigate whether NEDD8 can modify DNA-PKcs directly and its consequent effects on the activity of NHEJ repair pathway and cellular radiosensitivity.Contents:To determine whether DNA-PKcs can be neddylated;To identify the modified domain and the key site?s?;To identify the E3 ligase contributing to the DNA-PKcs neddylation;To illuminate whether and how neddylation regulates DNA-PKcs phosphorylation;To study the effects of DNA-PKcs neddylation on NHEJ efficiency of DNA DSBs repair and radiation sensitivity of cells.Methods:?1?SFB-NEDD8 wt and SFB-NEDD8 mutant with 76^thlysine mutation were constructed and overexpressed in 293T cells.24h after transfection,the cells were collected and followed by pull-down and Western blotting analysis to detect the neddylation of DNA-PKcs,Ku70 and Ku80;?2?He La cells were collected and lysed,and then DNA-PKcs or NEDD8 antibody was used for Co-IP to detect endogenous DNA-PKcs neddylation;?3?He La cells were pretreated with a final concentration of 3 ?M MLN4924 for 1h,and then IP was conducted with DNA-PKcs antibody to detect the inhibitory effect of MLN4924 on DNA-PKcs neddylation;?4?In vitro neddylation experiment was performed to further prove DNA-PKcs modification by NEDD8;?5?SFB-NEDD8 wt and NEDP1 or NEDP1 C163S were constructed and co-transfected into 293T cells,after 24h,cells were collected for pull-down and Western blotting to detect the modification of DNA-PKcs by NEDD8;?6?Flag-tagged DNA-PKcs truncations?A-H?vectors were constructed and overexpressed in 293T cells,followed by IP and Western blotting analysis to determine the position neddylated in DNA-PKcs;?7?The conserved lysines in the kinase domain of DNA-PKcs were mutated and overexpressed in 293T cells,followed by IP and mass spectrometry experiments to identify the key neddylated site;?8?Both K48 and K60 of NEDD8were mutated,and then overexpressed simultaneously with DNA-PKcs kinase domain in 293T cells to determine which lysis site of NEDD8 that DNA-PKcs neddylation is depended on;?9?DNA-PKcs kinase domain was overexpressed in 293T cells,after24h,cells were collected and lysed followed by immunoprecipitation-mass spectrometry assay,in combination of si RNA technology to confirm the E3 ligase contributing to DNA-PKcs neddylation;?10?NEDD8 was overexpressed in 293T cells,after 24h,cells were treated with 10 Gy irradiation.After 1h,cells were collected for pull-down and Western blotting analysis to investigate the association of DNA-PKcs neddylation with ionizing radiation exposure;?11?He La cells were pretreated with a final concentration of 3?M MLN4924 or DMSO.After 1h,the medium was replaced and cells were treated with different does?2Gy,4Gy and 8Gy?of irradiation,then cells were collected after 1h.Alternatively,cells were exposed to 4 Gy IR and collected at different time points?1?2?4h?.Then cells were lysed followed by Western blotting to detect the phosphorylation of DNA-PKcs;?12?Pretreated Hela with MLN4924 or DMSO for 1h,and then the medium was replaced.Cells were exposed to a final concentration of 30?g/ml VP16 for different time?1h,2h,4h,8h and 12h?were collected and then the DNA-PKcs phosphorylation was detected by Western blotting;?13?He La cells were transfected with si RNAs of UBA3,UBE2M and HUWE1.After 48h,cells were exposed to 4Gy and then collected and lysed to detect the phosphorylation of DNA-PKcs using Western blotting;?14?He La cells pretreated with MLN4924 or DMSO for 1h were irradiated?4Gy?or not.After 1h,cells were collected and the recruitment of DNA-PKcs at broken sites was detected by laser confocal imaging;?15?He La cells pretreated with MLN4924 or DMSO were performed 4Gy irradiation and collected at different time points?1h,2h and 4h?.Then the chromosome proteins were extracted to detect the recruitment of DNA-PKcs;?16?The cell cycle distribution of He La sh-NC and sh-HUWE1 cell lines at 4h and 8h after 4Gy irradiation was detected by flow cytometry;?17?The formation of?H2AX foci in He La sh-NC and sh-HUWE1 cell lines before and after 4Gy irradiation was analyzed by immune fluorescence staining;?18?The control si RNA or HUWE1si RNA or 53BP1 si RNA was transfected into 293T cells,and the repair system vectors of NHEJ were then transfected again after 24h.After another 24h,flow cytometry was performed to detect the repair efficiency of NHEJ;?19?Hela sh-NC and sh-HUWE1cells were seeded into 6-well plate followed by irradiation to analyze the sensitivity.Results:Through analysis of the gene-expression data from four biochips assays in the GEO database,which include the gene-expression profiles of 410 hunman lung cancers tissues,it was found that the expression of DNA-PKcs and NEDD8 was positively correlated in the tissues of human lung small cell cancers.Our experiment studies indicated that both the exogenous NEDD8 transfected and cellular endogenous NEDD8 could modify DNA-PKcs,and the NAE inhibitor MLN4924 could inhibit this modification.NEDD8 was also confirmed to modify DNA-PKcs in cell-free reaction assay.NEDP1 was found to be the de-neddylation enzyme to remove the neddylation modification of DNA-PKcs,wherase when the cells were transfected with the mutated NEDDP1,DNA-PKcs neddylation was not affected.This demonstrated that NEDP1 contributed for de-neddylation of DNA-PKcs.A set of DNA-PKcs truncated vectors were constructed and overexpressed in 293T cells,and it was found that DNA-PKcs kinase domain was mainly modified region.Further,we confirmed K4007 as the key modified site.The DNA-PKcs kinase domain was overexpressed in the cells,followed by mass spectroscopy analysis,in combination of the small RNA interference experiments,the HUWE1 was found to be the E3 ligases to catalyze the DNA-PKcs neddylation.Subsequently,we found that the interaction between HUWE1,DNA-PKcs and UBE2M were enhanced in the cells after exposure of ionizing radiation and DNA double-strand breaks induced.Simultaneously,DNA-PKcs neddylation was also increased,which was confirmed by co-immunoprecipitation.Finally,we studied the effects of neddylation on DNA-PKcs especially on its phosphorylation status and cellular radiosentivity.Inhibition of DNA-PKcs neddylation by MLN4924 or si RNAs of UBA3,UBE2M and HUWE1 impaired the phosphorylation of DNA-PKcs S2056 but not T2609.Furthermore,we found DNA-PKcs release from the broken DNA ends of chromatins was inhibited after MLN4924 treated.HUWE1 deletion led to decreased efficiency of non-homologous end joining activity of DNA DSBs repair.HUWE1 deleted cells were exhibited an increased blockage of G1 phase,and the radiation sensitivity of cells also increased dramatically.Conclusion:In the cellular response to ionizing radiation-induced DNA damage,HUWE1 neddylates the kinase domain of DNA-PKcs and promotes its phosphorylation on ser2056 and NHEJ repair efficiency in favour of cell survival.