Essential Role Of APE1 Acylation In Genome Stability Maintenance

DNA apurinic or pyrimidine site(AP)endonuclease-APE1 is the main protein that clears AP sites in the body,plays a central role in the response of cells to oxidative stress,and is widely involved in a variety of important metabolic processes in cells.Abnormal function of APE1 will cause DNA repair defects,which will lead to the instability of the genome and the occurrence of diseases.In severe cases,it will even lead to early embryonic death.It is precisely because of the important role of APE1 to maintain the stability of the genome that its activity and function must be extremely strictly regulated.Recent studies have shown that it shows that the activity of APE1 is regulated by a variety of post-translational modifications,including methylation,acylation,phosphorylation,and ubiquitination.Among them,the succinylation of APE1 protein currently only has mass spectrometry identification data,and its specific mechanism is not clear.Acetylation was found in previous studies to be one of its most important types,but its research mainly focused on the N-terminal,APE1 The acetylation modification function of the active region of DNA repair is still unclear.Therefore,it is of great significance to explore its acylation modification to explore the specific mechanism of APE1 acylation modification from basic science or clinical disease occurrence and development.Based on the mass spectrometry data reported in the previous period,this project constructed a series of point mutant recombinant proteins and mutant cell lines.The specific functions and related mechanisms of APE1 acylation modification were studied at the molecular and cellular levels.The specific conclusions as follows:1)Through in vivo and in vitro experiments,the in vivo succinylation of APE1 was detected,and the Lys125,Lys197 and Lys276 sites of APE1 were verified as important acylation sites.Among them,Lys125 and 276 are both acetylation sites and succinylation sites,and Lys197 is a succinylation site,and its acetylation modification is not obvious.2)Through in vitro cleavage activity experiments of point mutant proteins,it is found that the acylation modification of APE1 will affect the cleavage activity of its specific substrate.When Lys 125,197,276 position lysine is mutated to arginine(R),its AP cleavage activity is significantly reduced,and when the mutation is E(simulating continuous succinylation)or Q(simulating continuous acetylation),Its activity has been significantly restored.3)It is found that the acylation modification of APE1 can respond to DNA damage stress.Under different damage stress,when Lys 125,197,276 positions lysine is mutated to arginine,its survival rate decreases to varying degrees,and the cell damage signal is also significantly enhanced,while when the mutation is E or Q,Its tolerance to DNA damage pressure has been significantly improved.4)Identify 26 interacting proteins related to APE1 damage and stress by liquid phase mass spectrometry.Among them,there are 9 proteins related to acylation modification,and their protein abundance is more than 10 times that of the control group.It indicates that the acylation modification may respond to injury stress by affecting the interacting proteins of APE1.In summary,the results of this study have identified the main acylation modification sites of APE1,indicating that the acylation modification of APE1 can regulate enzyme cleavage activity,respond to different DNA damage stresses,and is likely to affect APE1 interacting proteins in a certain way Network,thereby regulating the protein involved in maintaining the stability of the cell genome.