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Real-Time Imaging of NADPH Oxidase Activity in Living Cells Using a Novel Fluorescent Protein Reporter

Production of reactive oxygen species (ROS) has been implicated in the pathology of many conditions, including cardiovascular, inflammatory and degenerative diseases, aging, muscular dystrophy, and muscle fatigue. NADPH oxidases (Nox) have recently gained attention as an important source of ROS invo...

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Autores principales: Pal, Rituraj, Basu Thakur, Poulami, Li, Shumin, Minard, Charles, Rodney, George G.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2013
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3660327/
https://www.ncbi.nlm.nih.gov/pubmed/23704967
http://dx.doi.org/10.1371/journal.pone.0063989
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author Pal, Rituraj
Basu Thakur, Poulami
Li, Shumin
Minard, Charles
Rodney, George G.
author_facet Pal, Rituraj
Basu Thakur, Poulami
Li, Shumin
Minard, Charles
Rodney, George G.
author_sort Pal, Rituraj
collection PubMed
description Production of reactive oxygen species (ROS) has been implicated in the pathology of many conditions, including cardiovascular, inflammatory and degenerative diseases, aging, muscular dystrophy, and muscle fatigue. NADPH oxidases (Nox) have recently gained attention as an important source of ROS involved in redox signaling. However, our knowledge of the source of ROS has been limited by the relatively impoverished array of tools available to study them and the limitations of all imaging probes to provide meaningful spatial resolution. By linking redox-sensitive GFP (roGFP) to the Nox organizer protein, p47(phox), we have developed a redox sensitive protein to specifically assess Nox activity (p47-roGFP). Stimulation of murine macrophages with endotoxin resulted in rapid, reversible oxidation of p47-roGFP. In murine skeletal muscle, both passive stretch and repetitive electrical stimulation resulted in oxidation of p47-roGFP. The oxidation of p47-roGFP in both macrophages and skeletal muscle was blocked by a Nox specific peptide inhibitor. Furthermore, expression of p47-roGFP in p47(phox) deficient cells restored Nox activity. As Nox has been linked to pathological redox signaling, our newly developed Nox biosensor will allow for the direct assessment of Nox activity and the development of therapeutic Nox inhibitors.
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spelling pubmed-36603272013-05-23 Real-Time Imaging of NADPH Oxidase Activity in Living Cells Using a Novel Fluorescent Protein Reporter Pal, Rituraj Basu Thakur, Poulami Li, Shumin Minard, Charles Rodney, George G. PLoS One Research Article Production of reactive oxygen species (ROS) has been implicated in the pathology of many conditions, including cardiovascular, inflammatory and degenerative diseases, aging, muscular dystrophy, and muscle fatigue. NADPH oxidases (Nox) have recently gained attention as an important source of ROS involved in redox signaling. However, our knowledge of the source of ROS has been limited by the relatively impoverished array of tools available to study them and the limitations of all imaging probes to provide meaningful spatial resolution. By linking redox-sensitive GFP (roGFP) to the Nox organizer protein, p47(phox), we have developed a redox sensitive protein to specifically assess Nox activity (p47-roGFP). Stimulation of murine macrophages with endotoxin resulted in rapid, reversible oxidation of p47-roGFP. In murine skeletal muscle, both passive stretch and repetitive electrical stimulation resulted in oxidation of p47-roGFP. The oxidation of p47-roGFP in both macrophages and skeletal muscle was blocked by a Nox specific peptide inhibitor. Furthermore, expression of p47-roGFP in p47(phox) deficient cells restored Nox activity. As Nox has been linked to pathological redox signaling, our newly developed Nox biosensor will allow for the direct assessment of Nox activity and the development of therapeutic Nox inhibitors. Public Library of Science 2013-05-21 /pmc/articles/PMC3660327/ /pubmed/23704967 http://dx.doi.org/10.1371/journal.pone.0063989 Text en © 2013 Pal et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Pal, Rituraj
Basu Thakur, Poulami
Li, Shumin
Minard, Charles
Rodney, George G.
Real-Time Imaging of NADPH Oxidase Activity in Living Cells Using a Novel Fluorescent Protein Reporter
title Real-Time Imaging of NADPH Oxidase Activity in Living Cells Using a Novel Fluorescent Protein Reporter
title_full Real-Time Imaging of NADPH Oxidase Activity in Living Cells Using a Novel Fluorescent Protein Reporter
title_fullStr Real-Time Imaging of NADPH Oxidase Activity in Living Cells Using a Novel Fluorescent Protein Reporter
title_full_unstemmed Real-Time Imaging of NADPH Oxidase Activity in Living Cells Using a Novel Fluorescent Protein Reporter
title_short Real-Time Imaging of NADPH Oxidase Activity in Living Cells Using a Novel Fluorescent Protein Reporter
title_sort real-time imaging of nadph oxidase activity in living cells using a novel fluorescent protein reporter
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3660327/
https://www.ncbi.nlm.nih.gov/pubmed/23704967
http://dx.doi.org/10.1371/journal.pone.0063989
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