{"title":"NADPH oxidase subunit p22phox: A marker of oxidase-dependent oxidative stress and target for stress suppression in nonphagocytic cells","authors":"Kei Miyano , Shuichiro Okamoto , Fumiya Ojima , Yasuhiro Takenouchi , Risa Yamamoto , Kimika Matsui , Misaki Azuhata , Mariko Inoue , Mizuho Kajikawa , Akira Yamauchi , Futoshi Kuribayashi , Shin-Ichiro Nishimatsu","doi":"10.1016/j.jim.2025.113850","DOIUrl":null,"url":null,"abstract":"<div><div>Reactive oxygen species (ROS)-producing NADPH oxidase (Nox) family proteins are involved in host defense. The overproduction of ROS leads to oxidative stress, which is associated with a myriad of diseases. The Nox subunit p22<sup><em>phox</em></sup> is essential for Nox1–4 activity, and p22<sup><em>phox</em></sup> and Nox2 proteins are mutually stabilized in phagocytic cells. This study investigated the suitability of p22<sup><em>phox</em></sup> protein as a marker of Nox activity. To avoid contamination by phagocytic p22<sup><em>phox</em></sup>, we developed global <em>Cybb</em> (encoding Nox2)-knockout mice and analyzed p22<sup><em>phox</em></sup> stability and the expression profiles of Nox proteins in lysates of various tissues. We found that consistent with Nox2 in phagocytic cells, p22<sup><em>phox</em></sup> protein was detected when Nox1–4 were coexpressed in nonphagocytic cells. Furthermore, p22<sup><em>phox</em></sup> protein degradation was suppressed by Nox1–4, suggesting that p22<sup><em>phox</em></sup> is a suitable marker of Nox family-dependent oxidative stress. Thus, we examined p22<sup><em>phox</em></sup> protein levels in tissue lysates prepared from <em>Cybb</em>-knockout mice to avoid the contamination of phagocytic p22<sup><em>phox</em></sup>. <em>Cybb</em>-knockout mice show moderately reduced p22<sup><em>phox</em></sup> protein levels in lung tissue, suggesting that Nox2 and other Nox family members stabilized p22<sup><em>phox</em></sup> protein. Paradoxically, this result implied that p22<sup><em>phox</em></sup> knockdown concurrently suppressed various Nox family-dependent oxidative stress mechanisms, and this was confirmed by the suppression of Nox family-dependent directed migration in p22<sup><em>phox</em></sup><em>-</em>knockdown A549 human lung epithelial cells. Therefore, p22<sup><em>phox</em></sup> not only served as a marker of Nox-dependent oxidative stress but also as a target to suppress this stress in various tissues and cells.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"539 ","pages":"Article 113850"},"PeriodicalIF":1.6000,"publicationDate":"2025-03-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Journal of immunological methods","FirstCategoryId":"3","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S002217592500050X","RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q4","JCRName":"BIOCHEMICAL RESEARCH METHODS","Score":null,"Total":0}
引用次数: 0
Abstract
Reactive oxygen species (ROS)-producing NADPH oxidase (Nox) family proteins are involved in host defense. The overproduction of ROS leads to oxidative stress, which is associated with a myriad of diseases. The Nox subunit p22phox is essential for Nox1–4 activity, and p22phox and Nox2 proteins are mutually stabilized in phagocytic cells. This study investigated the suitability of p22phox protein as a marker of Nox activity. To avoid contamination by phagocytic p22phox, we developed global Cybb (encoding Nox2)-knockout mice and analyzed p22phox stability and the expression profiles of Nox proteins in lysates of various tissues. We found that consistent with Nox2 in phagocytic cells, p22phox protein was detected when Nox1–4 were coexpressed in nonphagocytic cells. Furthermore, p22phox protein degradation was suppressed by Nox1–4, suggesting that p22phox is a suitable marker of Nox family-dependent oxidative stress. Thus, we examined p22phox protein levels in tissue lysates prepared from Cybb-knockout mice to avoid the contamination of phagocytic p22phox. Cybb-knockout mice show moderately reduced p22phox protein levels in lung tissue, suggesting that Nox2 and other Nox family members stabilized p22phox protein. Paradoxically, this result implied that p22phox knockdown concurrently suppressed various Nox family-dependent oxidative stress mechanisms, and this was confirmed by the suppression of Nox family-dependent directed migration in p22phox-knockdown A549 human lung epithelial cells. Therefore, p22phox not only served as a marker of Nox-dependent oxidative stress but also as a target to suppress this stress in various tissues and cells.
期刊介绍:
The Journal of Immunological Methods is devoted to covering techniques for: (1) Quantitating and detecting antibodies and/or antigens. (2) Purifying immunoglobulins, lymphokines and other molecules of the immune system. (3) Isolating antigens and other substances important in immunological processes. (4) Labelling antigens and antibodies. (5) Localizing antigens and/or antibodies in tissues and cells. (6) Detecting, and fractionating immunocompetent cells. (7) Assaying for cellular immunity. (8) Documenting cell-cell interactions. (9) Initiating immunity and unresponsiveness. (10) Transplanting tissues. (11) Studying items closely related to immunity such as complement, reticuloendothelial system and others. (12) Molecular techniques for studying immune cells and their receptors. (13) Imaging of the immune system. (14) Methods for production or their fragments in eukaryotic and prokaryotic cells.
In addition the journal will publish articles on novel methods for analysing the organization, structure and expression of genes for immunologically important molecules such as immunoglobulins, T cell receptors and accessory molecules involved in antigen recognition, processing and presentation. Submitted full length manuscripts should describe new methods of broad applicability to immunology and not simply the application of an established method to a particular substance - although papers describing such applications may be considered for publication as a short Technical Note. Review articles will also be published by the Journal of Immunological Methods. In general these manuscripts are by solicitation however anyone interested in submitting a review can contact the Reviews Editor and provide an outline of the proposed review.