Study On The Mechanism Of EIF4E2/GSK3β Regulating Redox Balance

Glycogen synthase kinase GSK3βplays a key role in the regulation of many signal pathways including cell cycle,inflammation and cell proliferation.Its target site usually contains pre-phosphorylation motif,and it can also regulate proline-directed serine/threonine phosphorylation.It has been revealed that the cap-binding protein eIF4E2directly binds to GSK3β,and only activates the proline-directed serine/threonine kinase activity of GSK3β;eIF4E2/GSK3βdirectly regulates the proline-directed serine/threonine phosphorylation of the transcription factor p53,including S33,S46,T81,S127,T150 and S315.The specific mechanism of inhibiting the eIF4E2/GSK3βpathway on cell viability needs to be further explored.This article mainly focuses on the upstream and downstream exploration of the eIF4E2/GSK3βpathway.The main research and results are as follows:（1）Inhibition of eIF4E2/GSK3βpathway down-regulateds the transcription and expression of disulfide bond reductase Trx1 protein in cells.（2）CHIP experiment found that p53 directly binds Trx1 promoter region.（3）Overexpression of wild-type p53(p53^WT),its mutant p53^6A（proline-directed serine/threonine sites targeted by eIF4E2/GSK3βwere mutated into alanine,mimicking non-phosphorylation）,and mutant p53^6D（proline-directed serine/threonine sites targeted by eIF4E2/GSK3βwere mutated to aspartic acid,simulating continuous phosphorylation）in p53-deficient cell lines,it was found that p53^6A and p53^WT significantly inhibit the transcription of Trx1,while p53^6D has no significant influence.（4）The detection of dual luciferase reporter gene found that p53 directly regulates Trx1gene transcription.p53^6A and p53^WT significantly inhibit Trx1 transcription.p53^6D also directly inhibits Trx1 transcription,but its effect is not as significant as that of p53^6A and p53^WT.（5）Inhibition of eIF4E2/GSK3βpathway down-regulates the transcription and expression of lipid hydroperoxidase GPX4.（6）The polypeptide e2-I can inhibit the interaction of eIF4E2/GSK3β.Ferrostatin-1,an ferroptosis inhibitor,can prevent e2-I from regulating the expression of GPX4 and Trx1.（7）e2-I treatment activates cellular reactive oxygen species and lipid peroxidation.（8）The combination of ferroptosis inducer erastin and e2-I will further peroxidize lipids.（9）Ferrostatin-1 cannot significantly reduce the reactive oxygen species induced by e2-I,nor can it restore the decrease in the number of colonies induced by e2-I.（10）LPS and doxorubincin stimulation can promote the interaction between eIF4E2 and GSK3β,which may depend on the ISG15 modification of eIF4E2.In summary,we found that eIF4E2/GSK3βmaintains the redox balance of cells by regulating the expression of Trx1 and GPX4.LPS and doxorubicin may activate the ISG15modification of eIF4E2,and then activate the eIF4E2/GSK3βpathway to regulate the redox imbalance caused by LPS and doxorubicin.Induction of cytoplasmic ROS production and inhibition of cell activity by e2-I did not depend on ferroptosis.Many chemotherapeutics kill cancer cells by relying on the activation of reactive oxygen species.Regulating the eIF4E2/GSK3βpathway may provide new ideas for tumor therapy by regulating the redox balance of cells.