The Inhibition And Molecular Mechanism Study Of IKIP Peptide In NF-κB Activation And Inflammation

The innate immune system provides the first line of defense against pathogens invasion.Activation of the innate immune system depends on recognition of conserved pathogen-asscocaited molecular patterns(PAMPs)by a set of pattern recognition receptors(PRRs).Toll like receptors(TLRs)can recognize bacterial lipopolysaccharide,lipopeptide,flagellin,viral nucleic acid as well as other antigenic components and initiate a series of signal pathways that lead to the production of pro-inflammatory cytokines and type I interferon,activate a strong immune response,and finally eliminate the pathogens.IKK complex plays an essential role in TLRs signal pathway,which contains two homologous catalytic subunits IKKa and IKKβ,and a regulatory subunit NEMO.IKK complex integrates signals from NF-κB activating stimulus,and catalyzes the phosphorylation of IκBs.IκBs are ubiquitinated and degraded by the proteasome,which in turn allow the nuclear translocation of NF-κB heterodimers that lead to the production of pro-inflammatory cytokines.However,aberrant or excessive proinflammatory cytokines production may cause tissue damage,even lead to cytokine storm that seriously threaten human health.Therefore,the production of proinflammatory cytokines is precisely regulated in vivo.A peptide derived from the NEMO binding domain(NBD)of IKKβ can block the association between IKKβ and NEMO and inhibit NF-κB activation.Furthermore,NBD peptide has shown strong therapeutic effects on several inflammation associated animal models.Whether other peptides target IKK complex and inhibit IKK activation need to be further explored.We previously demonstrated that IKIP interacted with IKKα/β and negatively regulated inflammation though N terminal region.In this study,we identified the function domain of IKIP,designed and synthesized peptides according to this sequence.And clarified the role and regulatory mechanism of the peptides in inflammation.Finally,we applied the peptides in inflammation associated animal model and rheumatoid arthritis(RA)to further determine the function of peptides in vivo.To further identify the function domain of IKIP,we constructed a series of mIKIP(mouse IKIP)deletion mutants.We found that IKIP FL(full length,1-374),IKIP(16374)and IKIP(31-374)could interact with IKKβ,and inhibit the NF-κB promoter activation and IKKβ phosphorylation,while IKIP(61-374),IKIP(76-374)and IKIP(91-374)had no effect.Taken together,these data demonstrated that the 31-60 aa sequence of IKIP is the function domain to inhibit inflammation.We designed peptide F0(31-45 aa)and F1(46-60 aa)from the 31-60 aa region of IKIP,and a negative control peptide F2(61-75 aa).All of the peptides had good cell permeability without detectable cytotoxicity.We next investigated the effect of the three peptides in inflammation regulation.We found that F1 peptide could inhibit NF-κB promoter activation and IKKβphosphorylation,while peptide F0 or F2 had no effect.Importantly,peptide F1 could also inhibit ligand stimulation-induced NF-κB activation and pro-inflammatory cytokines production,while F2 peptide had no effect.Taken together,these data demonstrated that F1 peptide is the active anti-inflammation peptide of IKIP.We next sought to determine the molecular mechanisms by which F1 peptide inhibited NF-κB activation.We found that F1 peptide interacted with IKKβ strongly while the interaction between IKKβ and peptide F0 or F2 peptide was marginal.Furthermore,F1 peptide competed with NEMO for binding to IKKβ.To determine the function of F1 peptide in vivo,we constructed lipopolysaccharide(LPS)-induced sepsis model and Zymosan-induced arthritis model.We found that F1 peptide could attenuate the pro-inflammatory cytokines production and pathological injury in inflammation associated animal model.To further extend the potential application of F1 peptide in treatment of human inflammatory diseases,we isolated synovial fibroblasts from synovial membrane of RA patients.F1 peptide could inhibit the pro-inflammatory cytokines production of synovial fibroblasts.In this study,we identified a peptide F1(46-60 aa of IKIP),which could suppress IKK activation and NF-κB targeted gene expression via disrupting the association of IKKβ and NEMO.Moreover,administration of F1 peptide attenuated LPS-induced sepsis and Zymosan-induced arthritis in vivo.F1 peptide also attenuated proinflammatory cytokines production in synovial fibroblasts from RA patients.Our findings provided a new therapeutic strategy for inflammatory diseases.Innovation:1.We found that peptide F1 negatively regulated NF-κB activation and inflammation.2.F1 peptide is a novel peptide-based inhibitor that targets IKK complex,and competes with NEMO for binding to IKKβ.3.Recently,inflammatory diseases have become commonly in clinic,and seriously affect the health and living quality of human being.We found that F1 peptide could attenuate the inflammatory reaction of sepsis and arthritis model.F1 peptide also attenuates pro-inflammatory cytokines production in synovial fibroblasts from RA patients.Our findings provide a new therapeutic strategy for the treatment and prevention of inflammatory diseases.