p67phox-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(265)NIVFVL(270), 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 xg; (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.

Automated summary

This study identified a novel mechanism of inhibiting NADPH oxidase activity using self-assembled peptides, which bind specifically to p67(phox) and deplete it, leading to the inhibition of oxidase activity.