Inhibition of NADPH oxidase activation by peptides mapping within the dehydrogenase region of Nox2-A "peptide walking" study

Iris Dahan, Shahar Molshanski-Mor, Edgar Pick*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

Abstract

In this study, the "peptide walking" approach was applied to the DH region of Nox2 (residues 288-570) with the purpose of identifying domains of functional importance in the assembly and/or catalytic function of the NADPH oxidase complex of phagocytes. Ninety-one overlapping 15- mer peptides were synthesized to cover the full length of the Nox2 DH region, and these were tested for the ability to interfere with the activation of the oxidase in vitro in two semirecombinant cell-free systems. The first consisted of phagocyte membranes p47 phox, p67 phox, and Rac1 and an amphiphile; the second was p47 phox- and amphiphile- free and contained prenylated Rac1. We identified 10 clusters of inhibitory peptides with IC 50 values of 10 μM, all of which were inhibitory, also in the absence of p47 phox. Based on the identification of residues shared by peptides in a particular cluster, we defined 10 functional domains in the Nox2 DH region. One domain corresponded to one FAD-binding subdomain, and four domains overlapped parts of three NADPH-binding subdomains. As expected, most inhibitory peptides acted only when added prior to the completion of oxidase assembly, but peptides associated with two NADPH-binding subdomains were also active after assembly. Kinetic analysis demonstrated that inhibition by peptides was not explained by competition for substrates (FAD, NADPH) but was of a more complex nature: noncompetitive with respect to FAD and uncompetitive with respect to NADPH. We conclude that oxidase-inhibitory peptides, in five out of 10 clusters identified, act by interfering with FAD- and NADPH-related redox reactions.

Original languageEnglish
Pages (from-to)501-515
Number of pages15
JournalJournal of Leukocyte Biology
Volume91
Issue number3
DOIs
StatePublished - Mar 2012

Keywords

  • Amphiphile
  • Cytochrome b558
  • P47
  • P67
  • Rac
  • Superoxide

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