Structure-function relationship in the interaction of mastoparan analogs with neutrophil NADPH oxidase.
Mastoparan, an amphiphilic cationic tetradecapeptide was previously shown to block activation of the NADPH oxidase in the cell-free system presumably by association with a cytosolic component/s of the enzyme. Blockade of oxidase activation was now demonstrated in the semirecombinant NADPH oxidase system. The structural basis of the inhibitory effect of MP on oxidase assembly was explored employing a variety of truncated and specifically substituted synthetic peptide analogs. The data indicated that an alpha helical fold, positive net charge, hydrophobicity and amphiphilicity were essential for the inhibitory potency and that peptide analogs below eleven residues were inactive. To identify the MP-binding oxidase subunit three different binding assays were carried out utilizing free or immobilized recombinant p47-phox, p67-phox, p40-phox and Rac1 in conjunction with immobilized MP or soluble (125)I-tyr-MP, respectively. The data implicated p67-phox as the main MP-binding component. The binding site on the p67-phox was localized to the 1-238 aminoterminal fragment of the molecule. NADPH oxidase activation supported by this fragment was inhibitable by MP. In addition, SH3 domains of p47-phox and p40-phox and the carboxyterminal SH3 domain of p67-phox exhibited a low affinity towards MP.
Tisch-Idelson
D
D
Department of Biochemistry, Tel Aviv University, Tel Aviv, Israel.
Fridkin
M
M
Wientjes
F
F
Aviram
I
I
D002474:Cell-Free System; D002850:Chromatography, Gel; D003600:Cytosol / Q000201:enzymology; D017353:Gene Deletion; D005982:Glutathione Transferase / Q000737:chemistry; D006801:Humans; D066298:In Vitro Techniques; D036341:Intercellular Signaling Peptides and Proteins; D007457:Iodine Radioisotopes; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009504:Neutrophils / Q000201:enzymology*; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D014862:Wasp Venoms / Q000737:chemistry / Q000494:pharmacology*
D036341:Intercellular Signaling Peptides and Proteins; D007457:Iodine Radioisotopes; D010455:Peptides; D010750:Phosphoproteins; D014862:Wasp Venoms; C085072:neutrophil cytosol factor 40K; C079789:neutrophil cytosol factor 67K; C037500:mastoparan; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; D005982:Glutathione Transferase