Inhibition of NADPH oxidase activation by peptides mapping within the dehydrogenase region of Nox2‐A “peptide walking” study

Journal of Leukocyte Biology: An Official Publication of the Reticuloendothelial Society

关键词: superoxide;    cytochrome b558 ;    p47phox ;    p67phox ;    Rac;    amphiphile;   

DOI  :  10.1189/jlb.1011507

学科分类：生理学

来源: Federation of American Societies for Experimental Biology

【 摘 要 】

Inthisstudy,the“peptidewalking”approachwasappliedtotheDHregionofNox2(residues288–570)withthepurposeofidentifyingdomainsoffunctionalimportanceintheassemblyand/orcatalyticfunctionoftheNADPHoxidasecomplexofphagocytes.Ninety‐oneoverlapping15‐merpeptidesweresynthesizedtocoverthefulllengthoftheNox2DHregion,andtheseweretestedfortheabilitytointerferewiththeactivationoftheoxidaseinvitrointwosemirecombinantcell‐freesystems.Thefirstconsistedofphagocytemembranesp47phox,p67phox,andRac1andanamphiphile;thesecondwasp47phox‐andamphiphile‐freeandcontainedprenylatedRac1.Weidentified10clustersofinhibitorypeptideswithIC50valuesof10μM,allofwhichwereinhibitory,alsointheabsenceofp47phox.Basedontheidentificationofresiduessharedbypeptidesinaparticularcluster,wedefined10functionaldomainsintheNox2DHregion.OnedomaincorrespondedtooneFAD‐bindingsubdomain,andfourdomainsoverlappedpartsofthreeNADPH‐bindingsubdomains.Asexpected,mostinhibitorypeptidesactedonlywhenaddedpriortothecompletionofoxidaseassembly,butpeptidesassociatedwithtwoNADPH‐bindingsubdomainswerealsoactiveafterassembly.Kineticanalysisdemonstratedthatinhibitionbypeptideswasnotexplainedbycompetitionforsubstrates(FAD,NADPH)butwasofamorecomplexnature:noncompetitivewithrespecttoFADanduncompetitivewithrespecttoNADPH.Weconcludethatoxidase‐inhibitorypeptides,infiveoutof10clustersidentified,actbyinterferingwithFAD‐andNADPH‐relatedredoxreactions...

【 授权许可】

CC BY