Cited by 150Neutrophil, Myeloperoxidase and Oxidative MechanismsRedox biology and oxidative stressNitric Oxide and Endothelin Effects

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Mapping sites of interaction of p47-phox and flavocytochrome b with random-sequence peptide phage display libraries.

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DeLeo, F R, Yu, L, Burritt, J B, Loetterle, L R, Bond, C W, Jesaitis, A J, Quinn, M T·1995·Proceedings of the National Academy of Sciences

DOI 10.1073/pnas.92.15.7110PMC PMC41481

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Abstract

During assembly of the phagocyte NADPH oxidase, cytosolic p47-phox translocates to the plasma membrane and binds to flavocytochrome b, and binding domains for p47-phox have been identified on the C-terminal tails of both flavocytochrome b subunits. In the present report, we further examine the interaction of these two oxidase components by using random-sequence peptide phage display library analysis. Screening p47-phox with the peptide libraries identified five potential sites of interaction with flavocytochrome b, including three previously reported regions of interaction and two additional regions of interaction of p47-phox with gp91-phox and p22-phox. The additional sites were mapped to a domain on the first predicted cytosolic loop of gp91-phox encompassing residues S86TRVRRQL93 and to a domain near the cytosolic C-terminal tail of gp91-phox encompassing residues F450EWFADLL457. The mapping also confirmed a previously reported binding domain on gp91-phox (E554SGPRGVHFIF564) and putative Src homology 3 domain binding sites on p22-phox (P156PRPP160 and G177GPPGGP183). To demonstrate that the additional regions identified were biologically significant, peptides mimicking the gp91-phox sequences F77LRGSSACCSTRVRRQL93 and E451WFADLLQLLESQ463 were synthesized and assayed for their ability to inhibit NADPH oxidase activity. These peptides had EC50 values of 1 microM and 230 microM, respectively, and inhibited activation when added prior to assembly but did not affect activity of the preassembled oxidase. Our data demonstrate the usefulness of phage display library analysis for the identification of biologically relevant sites of protein-protein interaction and show that the binding of p47-phox to flavocytochrome b involves multiple binding sites along the C-terminal tails of both gp91- and p22-phox and other regions of gp91-phox nearer to the N terminus.

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Authors: DeLeo, F. R., Yu, L., Burritt, J. B., Loetterle, L. R., Bond, C. W., Jesaitis, A. J., & Quinn, M. T.
Affiliations: Montana State University (US)
University of Washington (US)
ORCID: DeLeo, F R
Jesaitis, A J
Quinn, M T
Venue: Proceedings of the National Academy of Sciences
Publisher: National Academy of Sciences
Published: 1995-07-18
Volume: 92
Issue: 15
Pages: 7110-7114
ISSN: 0027-8424 (ISSN-L)
1091-6490
DOI: 10.1073/pnas.92.15.7110
PMC: PMC41481
OpenAlex: W2092234562
Cited by: 150
References: 40
Citations by year: 2026: 1 · 2025: 1 · 2024: 2 · 2023: 4 · 2021: 2 · 2020: 3 · 2019: 4 · 2018: 2 · 2017: 2 · 2016: 4 · 2015: 5 · 2014: 3 · 2013: 7 · 2012: 7
FWCI: 3.45
Type: journal article
Language: en
Open access: green
www.ncbi.nlm.nih.gov
Locations: Proceedings of the National Academy of Sciences (publishedVersion)
PubMed Central (publishedVersion)
PubMed (publishedVersion)
Funders: National Science Foundation
University of Missouri
National Institutes of Health (5r29ar040929-05)
National Institute of Allergy and Infectious Diseases (R01AI026711, AI26711)
National Institute of Arthritis and Musculoskeletal and Skin Diseases (AR40929)
Office of Experimental Program to Stimulate Competitive Research
Topics: Life Sciences › Immunology and Microbiology › Immunology › Neutrophil, Myeloperoxidase and Oxidative Mechanisms
Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Redox biology and oxidative stress
Health Sciences › Medicine › Physiology (medical) › Nitric Oxide and Endothelin Effects
Keywords: p47phox, NADPH oxidase, gp91phox, p22phox, protein-protein interaction, binding sites, phage display, Src homology 3 domain, NADPH oxidase inhibitors, phagocytes
MeSH terms: NADPH Oxidase 2, Binding Sites, Cytochrome b Group, Humans, Membrane Glycoproteins, NADH, NADPH Oxidoreductases, NADPH Dehydrogenase, Neutrophils, Peptide Fragments, Phosphoproteins, Protein Binding, Gene Library, NADPH Oxidases, Membrane Transport Proteins

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Mapping sites of interaction of p47-phox and flavocytochrome b with random-sequence peptide phage display libraries.

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