Interaction between p22phox and Nox4 in the endoplasmic reticulum suggests a unique mechanism of NADPH oxidase complex formation

• Published in: Free radical biology & medicine 2018-02, Vol.116, p.41-49
• Main Authors: Zana, Melinda, Péterfi, Zalán, Kovács, Hajnal A., Tóth, Zsuzsanna E., Enyedi, Balázs, Morel, Françoise, Paclet, Marie-Hélène, Donkó, Ágnes, Morand, Stanislas, Leto, Thomas L., Geiszt, Miklós
• Format: Article
• Language: English
• Subjects: Hydrogen peroxide; NADPH oxidase; Nox4; P22phox; Reactive oxygen species
• ISSN: 0891-5849; 1873-4596
• DOI: 10.1016/j.freeradbiomed.2017.12.031

The p22phox protein is an essential component of the phagocytic- and inner ear NADPH oxidases but its relationship to other Nox proteins is less clear. We have studied the role of p22phox in the TGF-β1-stimulated H2O2 production of primary human and murine fibroblasts. TGF-β1 induced H2O2 release of the examined cells, and the response was dependent on the expression of both Nox4 and p22phox. Interestingly, the p22phox protein was present in the absence of any detectable Nox/Duox expression, and the p22phox level was unaffected by TGF-β1. On the other hand, Nox4 expression was dependent on the presence of p22phox, establishing an asymmetrical relationship between the two proteins. Nox4 and p22phox proteins localized to the endoplasmic reticulum and their distribution was unaffected by TGF-β1. We used a chemically induced protein dimerization method to study the orientation of p22phox and Nox4 in the endoplasmic reticulum membrane. This technique is based on the rapamycin-mediated heterodimerization of the mammalian FRB domain with the FK506 binding protein. The results of these experiments suggest that the enzyme complex produces H2O2 into the lumen of the endoplasmic reticulum, indicating that Nox4 contributes to the development of the oxidative milieu within this organelle. [Display omitted] •The Nox4 protein expression is regulated by the presence of p22phox in primary fibroblasts.•The p22phox protein is stable in the absence of detectable Nox4 expression.•Nox4 and p22phox co-localize in the endoplasmic reticulum.•Orientation of the enzyme complex suggests H2O2 production into the ER lumen.