p67^phox-derived self-assembled peptides prevent Nox2 NADPH oxidase activation by an auto-inhibitory mechanism

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 ²⁶⁵NIVFVL²⁷⁰, exposed at either the N- or 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.