p67phox-derived self-assembled peptides prevent Nox2 NADPH oxidase activation by an auto-inhibitory mechanism

Abstract

Activation of the Nox2-dependent NADPH oxidase is the result of a conformational change in Nox2 induced by interaction with the cytosolic component p67 phox. In preliminary work we identified a cluster of overlapping 15-mer synthetic peptides, corresponding to p67 phox residues 259-279, which inhibited oxidase activity in an in vitro, cell-free assay, but the results did not point to a competitive mechanism. We recently identified an auto-inhibitory intramolecular bond in p67 phox , one extremity of which was located within the 259-279 sequence, and we hypothesized that inhibition by exogenous peptides might mimic intrinsic auto-inhibition. In this study, we found that: (i) progressive N-and C-terminal truncation of inhibitory p67 phox peptides, corresponding to residues 259-273 and 265-279, revealed that inhibitory ability correlated with the presence of residues 265 NIVFVL 270 , exposed at either the Nor C-termini of the peptides; (ii) inhibition of oxidase activity was associated exclusively with self-assembled peptides, which pelleted upon centrifugation at 12,000 ×g; (iii) self-assembled p67 phox peptides inhibited oxidase activity by specific binding of p67 phox and the ensuing depletion of this component, essential for interaction with Nox2; and (iv) peptides subjected to scrambling or reversing the order of residues in NIVFVL retained the propensity for self-assembly, oxidase inhibitory ability, and specific binding of p67 phox , indicating that the dominant parameter was the hydrophobic character of five of the six residues. This appears to be the first description of inhibition of oxidase activity by self-assembled peptides derived from an oxidase component, acting by an auto-inhibitory mechanism.

The molecular basis of Rac-GTP action—promoting binding of p67phoxto Nox2 by disengaging the β hairpin from downstream residues

“…Deletion of residues 259–279 disengaged the bond and generated a modified p67 phox which was bound selectively to a Nox2 peptide, corresponding to the sequence 369–383 (coined peptide 28) 35 . Peptides corresponding to residues 259–279 were found to act by a mechanism involving auto‐binding of p67 phox , mimicking the intramolecular bond 49 …”

“…35 Peptides corresponding to residues 259-279 were found to act by a mechanism involving auto-binding of p67 phox , mimicking the intramolecular bond. 49 We explored the molecular basis of the inhibitory action of peptides 106-120 and 181-195, prompted by the realization that the first coincided with the amino acid sequence of the β hairpin, and the second comprised residues K181 and R184, shown to be linked by intramolecular bonds to β hairpin residues Q115 and G113, respectively. 26,34…”

Structure, Activation, and Regulation of NOX2: At the Crossroad between the Innate Immunity and Oxidative Stress-Mediated Pathologies

“…Bechor et al identified p67 phox -derived self-assembled peptides corresponding to an auto-inhibitory intramolecular bond in p67 phox located within the 259-279 sequence [109]. In particular, purified peptides 265-270 and 265-279 inhibit NADPH oxidase activity with IC50 = 5.48 µM and 14.99 µM, respectively.…”

“…In particular, purified peptides 265-270 and 265-279 inhibit NADPH oxidase activity with IC50 = 5.48 µM and 14.99 µM, respectively. Inhibition of oxidase activity by self-assembled p67 phox peptides is mediated by binding p67 phox protein thus preventing its interaction with NOX2 [109].…”