The Endoplasmic Reticulum Chaperone Calnexin Is a NADPH Oxidase NOX4 Interacting Protein

Abstract

Within the family of NADPH oxidases, NOX4 is unique as it is predominantly localized in the endoplasmic reticulum, has constitutive activity, and generates hydrogen peroxide (H2O2). We hypothesize that these features are consequences of a so far unidentified NOX4-interacting protein. Two-dimensional blue native (BN) electrophorese combined with SDS-PAGE yielded NOX4 to reside in macromolecular complexes. Interacting proteins were screened by quantitative SILAC (stable isotope labeling of amino acids in cell culture) co-immunoprecipitation (Co-IP) in HEK293 cells stably overexpressing NOX4. By this technique, several interacting proteins were identified with calnexin showing the most robust interaction. Calnexin also resided in NOX4-containing complexes as demonstrated by complexome profiling from BN-PAGE. The calnexin NOX4 interaction could be confirmed by reverse Co-IP and proximity ligation assay, whereas NOX1, NOX2, or NOX5 did not interact with calnexin. Calnexin deficiency as studied in mouse embryonic fibroblasts from calnexin−/− mice or in response to calnexin shRNA reduced cellular NOX4 protein expression and reactive oxygen species formation. Our results suggest that endogenous NOX4 forms macromolecular complexes with calnexin, which are needed for the proper maturation, processing, and function of NOX4 in the endoplasmic reticulum.

Redox signals at theER–mitochondria interface control melanoma progression

“…ER stress was not observed, but our data showed that NOX4, which is upregulated in melanoma (Yamaura et al , ; Meitzler et al , ), is also a key player in ROS production following TMX knockdown. This is in agreement with a study showing that TMX3 is an interaction partner for NOX4 (Prior et al , ). These findings also indicate that TMX oxidoreductases serve as suppressors of NOX4 and thereby as cellular antioxidants, a concept that is in line with the findings reported in a recent study (Phan et al , ).…”

“…An alternative source of ROS, and thereby H 2 O 2 , within cells are the NADPH oxidases. NADPH oxidase 4 (NOX4) is highly upregulated in melanoma and absent in healthy skin (Yamaura et al , ; Meitzler et al , ) and the interaction between TMX proteins and NOX4 was previously reported, proposing the presence of NOX4 within the MAM domains (Prior et al , ). Hence, we examined the impact of NOX4 on cellular ROS production in TMX1‐silenced cells with the H 2 O 2 sensor HyPer.…”

The mitochondrial complexome of Arabidopsis thaliana

“…The resulting heat maps allow the identification of proteins forming part of protein complexes. Although introduced only relatively recently, this strategy has already added an additional degree of depth to the systematic identification of protein complexes in cells or subcellular fractions, albeit so far mainly in mammals (Heide et al ., ; Takabayashi et al ., ; Wessels et al ., ; De Almeida et al ., ; Huynen et al ., ; Müller et al ., ; Prior et al ., ; Wöhlbrand et al ., ).…”

NADPH oxidase 4 and its role in the cardiovascular system

“…In addition to the type of ROS generated by NOX4, its subcellular localization also influences various NOX4 functions, including enzyme activity and the activation of distinct downstream signaling pathways (8,9). However, the exact location of NOX4 remains largely debated, with reports positioning the enzyme in the endoplasmic reticulum, mitochondria, plasma membrane and nucleus (10,11). The reasons for these disparities may reflect the cell-specific differences in the functions of NOX4 in the different cell types studied, the fact that NOX4 localization might be transitory based on its interactions with certain targets (12) and/or the quality of research tools and approaches employed.…”