The Scaffold Protein AXIN1 Promotes Formation Of GCL Holoenzyme In Response To APAP-induced Oxidative Stress

As a multifunctional scaffold protein,AXIN1 interacts with various proteins.It has been reported that AXINI is involved in multiple signaling pathways to regulate embryonic development,cell fate,energy metabolism,etc.We previously discovered that Axinl liver-specific knockout(Axinl LKO)mice showed more severe liver injury than wild type(WT)mice after acetaminophen(APAP)treatment.Importantly,the activity of GCL,the rate-limiting enzyme in the glutathione(GSH)biosynthesis pathway,was lower in Axinl LKO mice accompanying with a reduction in the level of GSH.Thus,AXIN1 might protect liver against APAP-induced liver injury via promoting GSH synthesis.However,the underlying molecular mechanism contributing to this phenotype was unclear.In this thesis,we observe that GCL activity is lower with a reduction in the level of GSH in primary hepatocytes of Axinl LKO mice after APAP treatment,which comfirms that it is AXIN1 in the hepatocytes that plays an important role in the process.It has been indentified that the catalytic activity of GCL holoenzyme,which comprises a catalytic subunit GCLC and a regulatory subunit GCLM is much higher than free GCLC.We find that AXIN1 interacts with both subunits of GCL in HEK293T cells,and the interaction between the two subunits reduces in lentivirus-mediated AXINI knockdown cells.Furthermore,APAP not only promotes the interaction between GCLC and GCLM but also induces the interactions between AXIN1 and GCL subunits in liver.Studies in the mice with adeno-associated virus-mediated GCL overexpression further demonstrate that AXINI is vital for promoting the formation of GCL holoenzyme when treated with APAP These results indicate that AXIN1 protects liver against APAP-induced GSH depletion via promoting GCL holoenzyme formation by acting as a scaffold protein.Our findings reveal a new role of the multifunctional protein AXIN1 in response to oxidative stress,and may provide a potential therapeutic target for APAP-induced hepatotoxicity.