Mechanism Of DNA Damage Repair Protein Rad51 Ubiquitin Degraded By Autophagic Substrate P62 In Pathogenesis Of Helicobacter Pylori

Background and objective:According to statistics,gastric cancer ranks third in the global mortality rate of malignant tumors.There are many factors in the pathogenesis of gastric cancer.It has been established that Helicobacter pylori(Hp)infection plays a key role in the development of chronic non-atrophic gastritis to gastric cancer.Cytotoxin-associated protein(CagA)is one of the key virulence factors for Hp pathogenesis.At present,the exact pathogenic mechanism of Hp has not yet been elucidated.DNA double-strand breaks(DSBs)refer to the cleavage of DNA double strands at adjacent sites and are one of the most deadly forms of DNA damage.DSBs can cause cell genome instability and may eventually lead to cell malignant transformation.Our previous studies and recent foreign studies have found that Hp infection can induce DSBs in gastric mucosal epithelial cells.The precise repair of DSBs relies on DNA damage response(DDR)and homologous recombination(HR).Rad51 protein is a key protein for homologous recombination repair.It can be bound to the DNA site of excision modification under the recruitment and loading of BRCA2B,and participate in the process of homologous site search and DNA single-strand exchange.Our previous study found that Hp infection inhibits the expression of the key protein Rad51 in HR repair by inducing DSBs in gastric mucosal epithelial cells.Further studies have found that Hp infection promotes the occurrence of DSBs by inhibiting autophagy,and Rad51 plays an important role in autophagy to regulate DNA damage.However,the molecular mechanism of autophagy regulation of Rad51 and its role in Hp pathogenesis remains unclear.P62,also known as SQSTM1,is a multifunctional protein that,in addition to its autophagy receptor protein,is itself a selective substrate for autophagy.When autophagy occurs in cells,p62 protein is degraded;when autophagy activity is weakened or autophagy is deficient,p62 protein accumulates in cells,so p62 is one of the marker proteins that reflect autophagy activity.At the same time,p62 is an important regulatory molecule that links ubiquitinated proteins to autophagy,linking LC3 and ubiquitinated substrates,which are then integrated into autophagosomes and degraded in autophagosomes.P62 contains multiple domains,and its C-terminus contains a ubiquitin-binding domain(UBA domain).Its UBA sequence can selectively bind to polyubiquitinated proteins,and can directly act on the proteasome through the N-terminal UBL domain.The polyubiquitinated protein substrate is localized to the proteasome and degraded.Based on our previous research and previous literature reports,we hypothesized that the down-regulation of the DNA damage repair protein Rad51 caused by Hp infection is caused by p62-mediated ubiquitination.This study will investigate the effect of Hp infection and virulence factor CagA on p62 and Rad51,and further clarify the molecular regulation mechanism of autophagy substrate p62 to Rad51 during Hp infection.Materials and methods:?.The effect of Hp infection on p62 and Rad51 proteins in vitro and in vivo and the role of virulence factor CagA in it:1.Hp 7.13 was co-cultured with GES-1 and AGS cells in vitro.The expression of p62 and Rad51 under different infection conditions was detected by western blot.2.The animal model of C57BL/6 mice infected with HpSS1 was constructed.The colonization of Hp in the stomach of mice was detected by immunohistochemistry and fluorescence in situ hybridization.The changes of inflammatory factors in the stomach of mice were detected by real-time PCR.The expressions of p62 and Rad51in the gastric mucosa of mice were detected by immunohistochemistry and western blot.3.Hp7.13 and Hp7.13 CagA~-were co-cultured with GES-1 and AGS cells in vitro under the same infection conditions.The changes of p62 and Rad51 proteins were detected by western blot.?.Mechanisms of ubiquitous degradation of Rad51 protein by p62 in Hp infection:1.To investigate the effect of interfering p62 expression on Rad51 protein during the pathogenesis of Hp infection:p62 overexpression and knockdown plasmid were transfected into GES-1 and AGS cells,and Rad51 protein expression was detected by Western blot;Hp7.13 co-cultured with p62 knockdown GES-1 and AGS,and Rad51protein expression was detected by Western blot;an animal model of HpSS1 infected C57BL/6 mice was constructed to interfere with gastric mucus of mice.After the expression of p62 in membrane tissue,the expression of Rad51 protein was detected by immunohistochemistry and Western blot.2.To study whether there is interaction between p62 and Rad51 protein after Hp infection:Hp7.13 co-cultured with GES-1 and AGS cells,cell localization of p62 and Rad51 protein after infection was detected by immunofluorescence assay;Myc-p62and Flag-Rad51 plasmids were co-transfected into cells,and the interaction between p62 and Rad51 was detected by immunoprecipitation(Co-IP);String database predicted the interaction between p62 and Rad51 protein.3.Mechanisms of ubiquitous degradation of Rad51 protein by p62 in Hp infection:Protease inhibitor MG-132 pretreated AGS cells and co-cultured with Hp,Western blot was used to detect the expression of Rad51 protein;HA-Ub and Flag-Rad51 plasmids were co-transformed.The cells were stained and co-cultured with Hp.The effect of Hp infection on the ubiquitination level of Rad51 was detected by co-immunoprecipitation.The p62 protein was knocked down in AGS cells to detect the half-life of Rad51 protein.Myc-p62 and Flag-Rad51 were detected.The HA-Ub plasmid was co-transfected into GES-1 and AGS cells,and ubiquitination assay was used to detect whether p62 could cause ubiquitination degradation of Rad51.Results:?.The effect of Hp infection on p62 and Rad51 proteins in vitro and in vivo and the role of virulence factor CagA in it:Hp 7.13 standard strains infected AGS and GES-1 cells with different infections for 24 h,or infected cells with MOI=200 for different time.It was found that Hp infection in vitro resulted in increased expression of p62 protein and decreased expression of Rad51 protein.At the same time,in C57BL/6 mice infected with HpSS1 in vivo,it was also found that Hp infection could increase the expression of p62 protein,while decrease expression of Rad51 protein.Hp 7.13 and Hp 7.13 CagA~-were co-cultured with GES-1 and AGS cells under the same infection conditions in vitro.The expression of p62 and Rad51 protein was detected by western blot.It was found that the expression levels of p62 and Rad51protein were similar to those of the control group after deletion of CagA,the main factor of Hp.The above experiments suggest that Hp infection in vitro and in vivo can lead to accumulation of p62 protein and degradation of Rad51 protein,and CagA,the main virulence factor of Hp,is involved in this process.?.Mechanisms of ubiquitous degradation of Rad51 protein by p62 in Hp infection:The p62 overexpression plasmid and the knockdown plasmid were transfected into the cells.The expression level of Rad51 protein was significantly decreased when p62 was overexpressed,while the level of Rad51 protein was significantly increased when p62 was knocked down.At the same time of Hp infection in vivo and in vitro,the expression level of p62 protein was interfered.It was found that p62 protein was lower and Rad51 protein was higher in Hp infection.These results suggest that p62protein may be involved in the degradation of Rad51 protein during the infection of Hp in vivo and in vitro.We detected the localization of p62 and Rad51 proteins in cells by immunofluorescence assay,and found that there was co-localization between the two proteins during Hp infection.In addition,we found that exogenous p62 interacted with Rad51 by immunoprecipitation.Further,using String database,we found that p62 and Rad51 were linked by UBB protein,which was a ubiquitin protein.Treatment of the cells with the proteasome inhibitor MG-132 revealed significant inhibition of degradation of the Rad51 protein.Further,we co-transfected Flag-Rad51and HA-Ub into cells,and then co-cultured with Hp7.13 and Hp7.13 CagA~-respectively.It was found that there were obvious ubiquitination bands of Rad51protein in Hp 7.13 infected cells,while in Hp 7.13 CagA~-infected cells,the ubiquitination level of Rad51 protein decreased significantly.It indicated that Hp infection can induce the degradation of Rad51 ubiquitin,and the virulence factor CagA plays an important role.In addition,knockdown of p62 protein during Hp infection significantly prolonged the half-life of Rad51 protein,suggesting that the degradation of Rad51protein by p62 participated in the pathogenesis of Hp infection.Finally,we co-transfected Myc-p62,Flag-Rad51 and HA-Ub plasmids into cells.Ubiquitous experiments showed that the ubiquitination level of Rad51 protein increased significantly after transfection of p62 overexpression plasmid,indicating that p62 could ubiquitinate and degrade Rad51 protein.Conclusions:1.Hp infection can lead to accumulation of autophagy-related protein p62 and degradation of HR key protein Rad51,in which virulence factor CagA plays an important role.2.Hp infection mediates ubiquitination of Rad51,a key HR protein,by autophagy-related protein p62.