Abstract
NOX5 protein, one of the most active generators of reactive oxygen species (ROS), plays an important role in many processes, including regulation of cell growth, death and differentiation. Because of its central role in ROS generation, it needs to be tightly regulated to guarantee cellular homeostasis. Contrary to other members of NADPH-oxidases family, NOX5 has its own regulatory calcium-binding domain and thus could be activated directly by calcium ions. While several mechanisms of activation have been described, very little is known about the mechanisms that could prevent the overproduction of ROS by NOX5. In the present study using calorimetric methods and circular dichroism we found that oxidation of cysteine and methionine residues of NOX5 decreases binding of Ca2+ ions and perturbs both secondary and tertiary structure of protein. Our data strongly suggest that oxidation of calcium-binding domain of NOX5 could be implicated in its inactivation, serving as a possible defense mechanism against oxidative stress.
MeSH terms
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Amino Acid Sequence
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Calorimetry
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Calorimetry, Differential Scanning
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Chromatography, Liquid
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Circular Dichroism
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Cysteine / metabolism
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Homeostasis
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Humans
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Membrane Proteins / chemistry*
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Membrane Proteins / genetics
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Membrane Proteins / metabolism*
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Methionine / metabolism
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Molecular Sequence Data
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NADPH Oxidase 5
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NADPH Oxidases / chemistry*
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NADPH Oxidases / genetics
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NADPH Oxidases / metabolism*
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Oxidation-Reduction
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Protein Domains
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Reactive Oxygen Species / metabolism*
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Sequence Analysis, Protein
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Tandem Mass Spectrometry
Substances
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Membrane Proteins
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Reactive Oxygen Species
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Methionine
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NADPH Oxidase 5
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NADPH Oxidases
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NOX5 protein, human
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Cysteine
Grants and funding
The analysis of α-helical content and thermal denaturation of NOX5-EF as well as analysis of NOX5-EF oxidation level were supported by the Russian Science Foundation (grant #14-14-01152). Funding was also provided by European Union Seventh Framework Programme [FP7/2007-2013] Marie Curie Actions FP7-PEOPLE-2013-IIF#627524 (
http://ec.europa.eu/research/participants/portal/desktop/en/opportunities/fp7/calls/fp7-people-2013-iif.html) for data analysis. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.