Arginine Methylation Of Nrf2 And Its Mechanism Of The Function During The Antioxidative Response

The living organisms are often exposed to oxides and electrophilic substances from both endogenous and exogenous surroundings(e.g.,mitochondrial aerobic respiration,ultraviolet radiation,chemical reagent,etc).These stresses produce a large amount of reactive oxide species(ROS).Once excessive ROS is accumulated in the cells,it will lead to the failure of oxide removal,which causes an imbalance of the redox reaction,that is,the oxidative stress.At the cellular level,oxidative stress can cause oxidative damage,leading to cell death and apoptosis.Oxidative stress also can cause the chronic and acute inflammation,which may lead to cancer,diabetes,cardiovascular disease,neurodegenerative diseases,etc.As a defense mechanism,cells have developed a set of antioxidant systems to neutralize the excess ROS,thereby protecting cells and tissues from oxidative damage in body.In this process of protection,the cap'n'collar(CNC)family of transcription factors play important roles in resistance of oxidative and electrophilic stresses.Among the CNC family members,the NF-E2-related factor 2(Nrf2)is critical for regulating the antioxidant and phase ?enzymes through antioxidant response element(ARE)-mediated transactivation.The Keapl-Nrf2-ARE signaling pathway is believed to be the most important signaling pathway in the antioxidant system.At the normal physiological level,Nrf2 often binds with Keap1 protein,which mediates Nrf2 ubiquitination,keeping a low level of Nrf2 expression in the cytoplasm.When oxidative stress occurs,oxides combine with Keapl and inhibit the formation of the Nrf2-Keap1 complex.Then,Nrf2 is phosphorylated and transferred into the nucleus where Nrf2 binds to the ARE promoter element of the target genes(phase ? and the antioxidant genes)to activate their expression to resistant the oxidative stress.In recent years,accumulating evidence has shown that the activity of Nrf2 is controlled by a variety of post-translational modifications,including phosphorylation,ubiquitination,acetylation and sumoylation.The post-translational modifications of Nrf2 protein is closely related with its function.Studies in this field are helpful to reveal a new mechanism in the process of cell antioxidative response.In resent study,it is reported that the Nrf2 protein has multiple lysine acetylation sites mediated by p300/CBP.And some studies have indicated that the p300/CBP complex contains two kinds of arginine methylation transferase,PRMT1 and CARM1,as transcriptional activators.However,to date,there has been little information about the arginine methylation modification of Nrf2 protein and its function.By performing the in vitro methylation assay,we found that Nrf2 protein was methylated by PRMT1,but not CARM1.Meanwhile,the IP and GST-pull down experiments validated that Nrf2 interacted with PRMT1 and was methylated.Next,we performed mass spectrometry analysis of the methylated Nrf2,and found that the residue arginine 437 was methylated by PRMT1.In addition,we mutated arginine 437 to lysine(R437K)for the in vitro methylation analysis,and the results showed that the arginine 437 of Nrf2 was indeed the unique methylation site mediated by PRMT1.Through analyzing the structure domain of Nrf2 protein,we found that arginine 437 was located in the DNA binding domain of the protein.Thus,we speculated that the methylation of R437 in Nrf2 is likely to be related to its function of DNA binding.Bt using the real-time PCR analysis,we identified that the methylated Nrf2 promoted the expression of its target genes,HO-1,GCLM and TXNRD1.Moreover,the ChIP and EMSA assays demonstrated that,both knockdown of PRMT1 and overexpression of Nrf2 R437K mutant reduced the ability of Nrf2 binding to its target genes.In order to further explore the physiological function of methylated Nrf2,the antioxidant tBHP was used to induce the cells to produce oxidative stress.Under this model,we found that the methylation level of Nrf2 was increased in the process of antioxidative response,suggesting that the methylated Nrf2 played an important role in the antioxidative response.Furthermore,the cell viability assay and total GSH detection experiment indicated that in the process of cell antioxidative stress,the methylated Nrf2 was able to promote the antioxidative ability,improve the viability of the cells and protect cells from damage by oxidative stress.In conclusion,this study identifies a novel modification of Nrf2,and confirms that the arginine 437 of Nrf2 is modified by PRMT1.This modification enhances the antioxidative function of Nrf2 in cell antioxidative response.This study has unrivaled the preliminary molecule mechanism regarding the arginine methylation of Nrf2 on anti-oxidative stress.Data presented in this thesis provide an important theoretical basis for the development of potential drugs targeting the post-translational modifications of Nrf2 for the therapies of diseases caused by oxidative damage.