Grass Carp(Ctenopharyngodon Idella) NRF2 Up-regulates The Expression Of HO-1 Through Interaction With ATF4 To Alleviat ER Stress

The endoplasmic reticulum(ER)have good sense and respond to cellular stress(including metabolic imbalances and/or protein folding imbalances).For the first receptor to respond to ER stress,PERK,together with other proximal signaling molecules,initiates a transcriptional and translational regulation program,termed as the unfolded protein response(UPR).The result of persistent ER stress is the accumulation of reactive oxygen species that promotes a state of oxidative stress.PERK signaling coordinates the convergence of ER stress with oxidative stress signaling via the activation of the nuclear transcription factor erythroid 2-related factor 2(NRF2)and transcription factor 4(ATF4).As a number of the Cap ‘n' Collar(CNC)family,NRF2 contains a typical BRLZ domain at its c-terminal and regulates the downstream target gene heme oxygenase 1(HO-1)in response to ER stress.To study the role of CiNRF2 in ER stress,a grass carp(Ctenopharyngodon idella)NRF2 ORF was cloned and identified.The ORF(1782 bp)encodes a polypeptide of 593 amino acids and contains a well conserved DNA binding domain(BRLZ domain)by analyze.Phylogenetic tree analysis revealed that CiNRF2 had a closer evolutionary relationship with others fish NRF2,especially with Cyprinus carpio NRF2.After sustained stimulation with tunicamycin(TM),CiNRF2 was significantly up-regulated in CIK(C.idellus kidney)cells.To study the protective mechanism of fish NRF2 signaling pathway in ER stress,the sequences of CiNRF2 and CiATF4(grass carp ATF4)were separately subcloned into the expression vectors pEGFP and pCMV-Flag for co-immunoprecipitation and GST-Pulldown assays.These assays indicated that CiNRF2 can combine with CiATF4.In order to study the molecular mechanism of interaction,CO-IP and GST-Pulldown analysis were performed after different structures were constructed.It was found that the ? Neh2 domain might be related to the interaction of Ci ATF4.Meanwhile,we cloned grass carp HO-1 promoter sequence and constructed recombinant plasmid of pGL3-HO-1.In addition,recombinant plasmid of pGL3-HO-1 was co-transfected into grass carp ovary cells(COs)with pcDNA3.1-CiNRF2 or pcDNA3.1-CiATF4,respectively.The results showed that both CiNRF2 and CiATF4 extremely significantly increased the luciferase activity of pGL3-HO-1.At the same time,only the Neh4,5 domain could enhance the luciferase activity of pGL3-HO-1 by transfecting different structures separately.Finally,CCK-8 experiments showed that CiNRF2 could enhance cell viability by up-regulating the activity of HO-1.