P62/Keap1-Nrf2 Pathway Mediated Autophagy Promotes Antioxidant Stress In Shellfish

Mussels are an important part of the aquatic biological community,which of biomass is dominant among benthic animals,and is the primary consumer of aquatic ecosystems.Microcystins(MC)produce by cyanobacterial blooms seriously affect the survival of mussels.Mussels can quickly accumulate MC in the body through direct feeding activities,which produce reactive oxygen species(ROS)to cause the imbalance of oxidation system,and result in cell damage.Selective autophagy and the Keap1-Nrf2 pathway are the main stress response pathways.ROS can induce autophagy and activate the Keap1-Nrf2 pathways to alleviate cell oxidative damage through multiple signal pathways.The activation pathways of Nrf2 are divided into classical and non-classical pathways.Under stress conditions,the conformation changes of Keap1 is modified,so that Nrf2 enters the nucleus and forms heterodimer with small Mafs,and induce the antioxidant enzyme function of Nrf2 target regulatory region,thereby protecting cells and tissues from oxidative damage,which is the classic pathway of Nrf2 activation.P62 is the adaptor of selective autophagy,interacts with ubiquitin connexin and shutters them to proteasome or lysosome,and participates in the degradation of ubiquitin-proteasome and autophagy-lysosome.Its expression level is usually inversely proportional to autophagy.In mammals,the p62 protein acts as a cellular integration point that coordinates autophagy and the Keap1-Nrf2 signaling pathways during oxidative stress,and protects cells from damage by forming a p62/Keap1-Nrf2 positive feedback loop to mediate autophagy up-regulating antioxidant responsive element(ARE)detoxification enzymes,which is the non-classical pathway of Nrf2 activation.In the paper,the bivalves were exposed to MC environment as the research object,the identification and functional of Cpp62 were analyzed,the relationship between p62 and autophagy,and the regulatory relationship between p62 and Keap1-Nrf2 pathway was clarified in bivalves,to explore the innate immune function of shellfish the p62/Keap1-Nrf2 pathways reducing Microcystis toxicity.1.The expression of Nrf2 mRNA and protein were significantly increased after MC-induced 24 h,and MC inhibited significantly that expression after knockdown of Nrf2,whereas that expression was significantly enhanced after knockdown CpKeap1a 96 h by the experiments of q RT-PCR,western blot,and immunohistochemical.MC promoted the activities of GOT,GPT,and ARE-driven enzymes significantly.MC upregulated the activity of Cp NQO1 promoter in a dose-dependent manner,and also promoted Cp Nrf2 to up-regulate that activity.2.The expression vectors of pGEX-4T-1-CpKeap1a,p ET30a-Keap1 b,and p ET-32a-Nrf2 were constructed,to purify GST-CpKeap1a and HIS-Cp Keap1 b protein by SDS-PAGE,and their concentrations were 0.338 mg/m L and 0.22 mg/m L,respectively.GST-pulldown results showed that CpKeap1a and Cp Keap1 b form a heterodimer,and co-immunoprecipitation experiments showed that CpKeap1a and Cp Keap1 b formed homodimers or heterodimers.3.These fragments of p62,LC3,and Bcl-2 were screened from C.plicata hemocyte transcriptome library,specific primers were designed,and the full-length c DNA sequences of Cpp62,CpLC3,and CpBcl-2 were cloned using RACE PCR.The full-length sequence of Cpp62 c DNA is 2484 bp,with 88 bp 5’UTR sequence,1247 bp 3’UTR sequence,and the ORF sequence of 1149 bp,encoding 382 amino acids,including the highly conserved p62-PB1 domain(3-90aa),ZZ chain domain(102-148aa)and UBA domain,predicted to have LIR domain,but no KIR domain.and contained a poly(A)tail,and its molecular weight was 42.485 k Da by mass spectrometry,the theoretical isoelectric point(PI)is 5.98.The full-length sequence of CpLC3 c DNA is 2151 bp with 64 bp 5’UTR sequence,1721 bp 3’UTR sequence,366 bp ORF,121 amino acids was encoded,containing Ub1＿ATG8＿MAP1LC3domain,and a poly(A)tail,which molecular weight is 14.10 k Da,and PI is 9.39.CpBcl-2 c DNA sequence is 760 bp with 93 bp 5’UTR sequence,132 bp 3’UTR sequence,the ORF sequence is 535 bp,to encode 177 aa,including the like superfamily domain of Bcl-2(44-145 aa),which molecular weight is 20.17 k Da.4.After MC-induced,Cpp62 expression levels were significantly up-regulated in the hepatopancreas and kidney of C.plicata,which was down-regulated after Cp Nrf2 knockdown,whereas that was up-regulated after knockdown CpKeap1a at5.24 h.The results showed that Keap1-Nrf2 pathway regulated Cpp62 relative expression level under MC-stimulated.6.The promoter sequence of p62 from C.plicata was cloned and analyzed,containing multiple binding elements,such as Nrf2,Maf G,Mafk,Nrf2-Maf G dimer,and ARE-elements.Electrophoretic mobility shift assay(EMSA)results showed that Cp Nrf2 protein had a blocking effect on Cpp62 promoter.The results of the luciferase reporter gene showed that Cp Nrf2 upregulated Cpp62-ARE luciferase activity in a concentration-dependent manner.However,MC inhibited Cp Nrf2 upregulated Cpp62-ARE activity,and the activity of Cpp62-ARE-mut was inhibited by Cp Nrf2.7.Synthetic ds RNA of p62 from Cristaria plicata(Cpp62-ds RNA),the interference efficiency of Cpp62-ds RNA was 64.7% and 58.6% at 24 and 48 h,respectively,indicating that Cpp62-ds RNA sequence has interference efficiency.The relative levels of Cp Nrf2 and Cp NQO1 were significantly down-regulated in Cpp62-ds RNA group.Cpp62 up-regulated the luciferase activity of Cp NQO1 and Cpp62 promoters in dose-dependent,whereas MC inhibited Cpp62 to promote Cpp62 promoter activity,and Cpp62 also inhibited the activity of Cpp62 promoter with ARE element mutation.8.Cpp62 did not bind to CpKeap1a in vitro.Dual-luciferase reports showed that Cpkeap1 a and Cpkeap1 b did not affect Cpp62 to up-regulate the of Cp NQO1-ARE and Cpp62-ARE activity.9.Constructed the autophagy inhibitor(3-MA)and autophagy activator(Rap)models of shellfish,the results showed that the dynamic changes of autophagy affected p62 expression level.TEM result showed that MC induced autophagy in C.plicata,and the number of autophagosomes decreased and autophagosomes disappeared in Cpp62 ds RNA group,which indicated that Cpp62 mediated the formation of autophagosomes in C.plicata,and there was Cpp62-dependent autophagy in shellfish.10.CpLC3 is expressed in all tissues of the experiment,the immunological double-fluorescence localization investigation revealed that the distribution of CpLC3 protein,Cpp62,and CpKeap1a protein in the cell was overlapped under MC stress.The expression of CpLC3 was up-regulated in 3-MA group,and MC significantly increased CpLC3 mRNA level.The results indicated that autophagyand oxidative stress-regulated CpLC3 relative expression.11.When autophagy was defective,the expression level of CpBcl-2 mRNA was significantly decreased,which was decreased when autophagy occurs.The results showed that 3-MA promoted apoptosis,and the apoptosis rate of Cpp62-ds RNA group was higher than the control group.The results showed that autophagy produced by MC-induced inhibited cell apoptosis.