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Molecular Insights of p47(phox) Phosphorylation Dynamics in the Regulation of NADPH Oxidase Activation and Superoxide Production
Phagocyte superoxide production by a multicomponent NADPH oxidase is important in host defense against microbial invasion. However inappropriate NADPH oxidase activation causes inflammation. Endothelial cells express NADPH oxidase and endothelial oxidative stress due to prolonged NADPH oxidase activ...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
American Society for Biochemistry and Molecular Biology
2014
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4132782/ https://www.ncbi.nlm.nih.gov/pubmed/24970888 http://dx.doi.org/10.1074/jbc.M114.561159 |
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author | Meijles, Daniel N. Fan, Lampson M. Howlin, Brendan J. Li, Jian-Mei |
author_facet | Meijles, Daniel N. Fan, Lampson M. Howlin, Brendan J. Li, Jian-Mei |
author_sort | Meijles, Daniel N. |
collection | PubMed |
description | Phagocyte superoxide production by a multicomponent NADPH oxidase is important in host defense against microbial invasion. However inappropriate NADPH oxidase activation causes inflammation. Endothelial cells express NADPH oxidase and endothelial oxidative stress due to prolonged NADPH oxidase activation predisposes many diseases. Discovering the mechanism of NADPH oxidase activation is essential for developing novel treatment of these diseases. The p47(phox) is a key regulatory subunit of NADPH oxidase; however, due to the lack of full protein structural information, the mechanistic insight of p47(phox) phosphorylation in NADPH oxidase activation remains incomplete. Based on crystal structures of three functional domains, we generated a computational structural model of the full p47(phox) protein. Using a combination of in silico phosphorylation, molecular dynamics simulation and protein/protein docking, we discovered that the C-terminal tail of p47(phox) is critical for stabilizing its autoinhibited structure. Ser-379 phosphorylation disrupts H-bonds that link the C-terminal tail to the autoinhibitory region (AIR) and the tandem Src homology 3 (SH3) domains, allowing the AIR to undergo phosphorylation to expose the SH3 pocket for p22(phox) binding. These findings were confirmed by site-directed mutagenesis and gene transfection of p47(phox−/−) coronary microvascular cells. Compared with wild-type p47(phox) cDNA transfected cells, the single mutation of S379A completely blocked p47(phox) membrane translocation, binding to p22(phox) and endothelial O(2)(⨪) production in response to acute stimulation of PKC. p47(phox) C-terminal tail plays a key role in stabilizing intramolecular interactions at rest. Ser-379 phosphorylation is a molecular switch which initiates p47(phox) conformational changes and NADPH oxidase-dependent superoxide production by cells. |
format | Online Article Text |
id | pubmed-4132782 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2014 |
publisher | American Society for Biochemistry and Molecular Biology |
record_format | MEDLINE/PubMed |
spelling | pubmed-41327822014-08-19 Molecular Insights of p47(phox) Phosphorylation Dynamics in the Regulation of NADPH Oxidase Activation and Superoxide Production Meijles, Daniel N. Fan, Lampson M. Howlin, Brendan J. Li, Jian-Mei J Biol Chem Computational Biology Phagocyte superoxide production by a multicomponent NADPH oxidase is important in host defense against microbial invasion. However inappropriate NADPH oxidase activation causes inflammation. Endothelial cells express NADPH oxidase and endothelial oxidative stress due to prolonged NADPH oxidase activation predisposes many diseases. Discovering the mechanism of NADPH oxidase activation is essential for developing novel treatment of these diseases. The p47(phox) is a key regulatory subunit of NADPH oxidase; however, due to the lack of full protein structural information, the mechanistic insight of p47(phox) phosphorylation in NADPH oxidase activation remains incomplete. Based on crystal structures of three functional domains, we generated a computational structural model of the full p47(phox) protein. Using a combination of in silico phosphorylation, molecular dynamics simulation and protein/protein docking, we discovered that the C-terminal tail of p47(phox) is critical for stabilizing its autoinhibited structure. Ser-379 phosphorylation disrupts H-bonds that link the C-terminal tail to the autoinhibitory region (AIR) and the tandem Src homology 3 (SH3) domains, allowing the AIR to undergo phosphorylation to expose the SH3 pocket for p22(phox) binding. These findings were confirmed by site-directed mutagenesis and gene transfection of p47(phox−/−) coronary microvascular cells. Compared with wild-type p47(phox) cDNA transfected cells, the single mutation of S379A completely blocked p47(phox) membrane translocation, binding to p22(phox) and endothelial O(2)(⨪) production in response to acute stimulation of PKC. p47(phox) C-terminal tail plays a key role in stabilizing intramolecular interactions at rest. Ser-379 phosphorylation is a molecular switch which initiates p47(phox) conformational changes and NADPH oxidase-dependent superoxide production by cells. American Society for Biochemistry and Molecular Biology 2014-08-15 2014-06-26 /pmc/articles/PMC4132782/ /pubmed/24970888 http://dx.doi.org/10.1074/jbc.M114.561159 Text en © 2014 by The American Society for Biochemistry and Molecular Biology, Inc. Author's Choice—Final version full access. Creative Commons Attribution Unported License (http://creativecommons.org/licenses/by/3.0/) applies to Author Choice Articles |
spellingShingle | Computational Biology Meijles, Daniel N. Fan, Lampson M. Howlin, Brendan J. Li, Jian-Mei Molecular Insights of p47(phox) Phosphorylation Dynamics in the Regulation of NADPH Oxidase Activation and Superoxide Production |
title | Molecular Insights of p47(phox) Phosphorylation Dynamics in the Regulation of NADPH Oxidase Activation and Superoxide Production |
title_full | Molecular Insights of p47(phox) Phosphorylation Dynamics in the Regulation of NADPH Oxidase Activation and Superoxide Production |
title_fullStr | Molecular Insights of p47(phox) Phosphorylation Dynamics in the Regulation of NADPH Oxidase Activation and Superoxide Production |
title_full_unstemmed | Molecular Insights of p47(phox) Phosphorylation Dynamics in the Regulation of NADPH Oxidase Activation and Superoxide Production |
title_short | Molecular Insights of p47(phox) Phosphorylation Dynamics in the Regulation of NADPH Oxidase Activation and Superoxide Production |
title_sort | molecular insights of p47(phox) phosphorylation dynamics in the regulation of nadph oxidase activation and superoxide production |
topic | Computational Biology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4132782/ https://www.ncbi.nlm.nih.gov/pubmed/24970888 http://dx.doi.org/10.1074/jbc.M114.561159 |
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