Regulation of Derlin-1-mediated degradation of NADPH oxidase partner p22phox by thiol modification

The transmembrane protein p22phox heterodimerizes with NADPH oxidase (Nox) 1–4 and is essential for the reactive oxygen species-producing capacity of oxidases. Missense mutations in the p22phox gene prevent the formation of phagocytic Nox2-based oxidase, which contributes to host defense. This results in chronic granulomatous disease (CGD), a severe primary immunodeficiency syndrome. In this study, we characterized missense mutations in p22phox (L51Q, L52P, E53V, and P55R) in the A22° type (wherein the p22phox protein is undetectable) of CGD. We demonstrated that these substitutions enhanced the degradation of the p22phox protein in the endoplasmic reticulum (ER) and the binding of p22phox to Derlin-1, a key component of ER-associated degradation (ERAD). Therefore, the L51-L52-E53-P55 sequence is responsible for protein stability in the ER. We observed that the oxidation of the thiol group of Cys-50, which is adjacent to the L51-L52-E53-P55 sequence, suppressed p22phox degradation. However, the suppression effect was markedly attenuated by the serine substitution of Cys-50. Blocking the free thiol of Cys-50 by alkylation or C50S substitution promoted the association of p22phox with Derlin-1. Derlin-1 depletion partially suppressed the degradation of p22phox mutant proteins. Furthermore, heterodimerization with p22phox (C50S) induced rapid degradation of not only Nox2 but also nonphagocytic Nox4 protein, which is responsible for redox signaling. Thus, the redox-sensitive Cys-50 appears to determine whether p22phox becomes a target for degradation by the ERAD system through its interaction with Derlin-1.


Introduction
The NADPH oxidase (Nox) family of enzymes produces reactive oxygen species (ROS) [1][2][3][4][5]. This family participates in variety biological functions, including host defense [1], signal transduction [6], otoconia synthesis [7], and hormone synthesis [8]. The human Nox family comprises seven members (Nox1-5, Duox1, and Duox2). Among the Nox family members, Nox2 (a.k.a. gp91 phox ) is the prototype and is expressed abundantly in professional phagocytes (e.g., neutrophils and macrophages), where it contributes to host defense by generating substantial quantities of superoxide. The superoxide generated is the precursor for other ROS (highly reactive), including hydrogen peroxide and hydroxyl radicals, which are involved in bacterial killing. Genetic defects in Nox2 (encoded by the X-linked CYBB gene) lead to chronic granulomatous disease (CGD), which is characterized by recurrent life-threatening bacterial and fungal infections [9].
Nonphagocytic Nox4 oxidase is expressed abundantly in endothelial cells (ECs) of blood vessels [18] and contributes to redox signaling, leading to changes in physiological processes, such as angiogenesis [19]. Unlike Nox2, Nox4 is primarily localized in the ER, where it also interacts with p22 phox [20][21][22]. The presence of p22 phox is also required for the detection of Nox4 protein. We previously reported that in transformed ECs, Nox4 protein levels are attenuated by the hypoxia-induced reduction of p22 phox mRNA and protein levels [23]. In addition, Nox4 was undetectable in an animal model expressing the p22 phox (Y121H) mutant protein with reduced protein expression instead of wild-type p22 phox [22,24].
The heterodimerization with p22 phox is indispensable for the localization of Nox2 in the phagocyte/plasma membrane. In addition, p22 phox functions as an anchor for the soluble cytosolic activating protein p47 phox , forming an active complex. The formation of a complex with Nox2 occurs through the interaction between p47 phox and p22 phox , because p47 phox forms a ternary complex with other activating proteins-p67 phox and p40 phox . A missense mutation in CYBA (p22 phox gene), which results in an amino acid substitution of glutamine for proline-156, impairs the binding of p22 phox to p47 phox [25,26]. Because the expression of p22 phox is responsible for Nox2 localization and activation, genetic defects and missense mutations in p22 phox also cause CGD.
The interaction with p22 phox is also required for Nox4 activity [20]. The activity of Nox4 is independent of the presence of Nox-activating proteins, such as p47 phox and p67 phox . The amount of ROS generated by Nox4 is proportional to the expression levels of Nox4-p22 phox . A switch for activating Nox2 is turned off by the dissociation of p22 phox and the soluble cytosolic activating protein p47 phox , whereas a switch for Nox4 activity is not readily turned off. To the best of our knowledge, a switch mechanism for the enzymatic activity of Nox4 has not been proposed yet.
A missense mutation in p22 phox (P156Q) has been functionally characterized [25,26]. The mutated proteins are unable to bind to p47 phox [25,26]. Thus, Pro-156 is considered to be responsible for the activation of Nox2. The mutational hotspot located in CYBA exon 3 in the A22 • type (wherein the p22 phox protein is undetectable) of CGD exhibits missense mutations in p22 phox (L51Q, L52P, E53V, and P55R) [16]; however, to the best of our knowledge, these mutations have not been characterized yet. In the present study, we characterized missense mutations in p22 phox (L51Q, L52P, E53V, and P55R) and demonstrated that these amino acid substitutions promote the degradation of p22 phox protein in the ER. Interestingly, all of the mutant proteins strongly promoted the binding of p22 phox to Derlin-1, a key component of the ERAD system [27][28][29][30]. These findings suggest that these amino acids (Leu-51, Leu-52, Glu-53, and Pro-55) are responsible for the stability of p22 phox protein in the ER. Furthermore, the L52P and E53V substitutions impaired the binding of p22 phox to Nox2. Thus, the L 51 -L 52 -E 53 -P 55 sequence is involved in Nox2-based oxidase activity through a mechanism different from that of Pro-156.
We further demonstrated that the stability of the p22 phox protein is regulated by redox-sensitive Cys-50, which is adjacent to the L 51 -L 52 -E 53 -P 55 sequence, in a thiol oxidation-dependent manner. A C50S substitution results in decreased protein stability. Moreover, blocking the free thiol of Cys-50 by alkylation or C50S substitution promoted the association of p22 phox and Derlin-1. The Nox2 and Nox4 proteins form a complex with p22 phox (C50S) and are rapidly degraded. Thus, the Cterminal region adjacent to Cys-50 (amino acids 50-55, including Cys-50) appears to be responsible for the stability of p22 phox and its partners, Nox2 and Nox4. Because the activity and stability of Nox4 are dependent on the presence of p22 phox [23], we propose that p22 phox degradation resulting from the modification of Cys-50 thiol group is a switch that turns off Nox4 activity.

Cells, cell culture, and plasmid transfection
The CHO-K1 or HeLa cells were cultured as described previously [31,33]. The plasmids were transfected into CHO-K1 or HeLa cells as previously described [31,33].

Immunofluorescence microscopy
Immunofluorescence microscopy was performed as described previously [21]. Briefly, to stain p22 phox -Myc, Derlin-1-FLAG and PDI (ER marker), plasmid-transfected CHO-K1 cells grown on coverslips were fixed for 15 min in 4% formaldehyde at room temperature and then for 10 min in ice-cold 100% methanol at − 20 • C, followed by permeabilization for 60 min in 0.3% Triton X-100 in PBS with 5% bovine serum albumin (BSA). The samples were incubated overnight at 4 • C with the indicated primary antibodies in PBS with 1% BSA and 0.3% Triton X-100; subsequently, these samples were incubated for 1-2 h at room temperature with secondary antibodies in PBS with 1% BSA and 0.3% Triton X-100. Mouse monoclonal antibodies against p22 phox (CS9) or Myc tag (9E10) were used to detect p22 phox (green), mouse monoclonal antibodies against FLAG (1E6) was used to detect Derlin-1-FLAG (magenta), a rabbit monoclonal antibody against PDI (C81H6) was used to detect the ER marker PDI (magenta) and Hoechst staining was used to detect cell nuclei (blue).

Assay of O 2 − or H 2 O 2 production
The production of O 2 − by cells expressing Nox2 was assayed using Diogenes-luminol solution as described previously [31]. Briefly, the transfected cells (7 × 10 5 cells in 6-well plates) were cultured for 24 h and harvested by incubation with trypsin/ethylenediaminetetra-acetic acid. After being washed with PBS, the cells were suspended at a density of 7 × 10 5 cells per 250 μl PBS plus 10 μl Diogenes-luminol solution.
The cells were treated with 200 ng/ml phorbol 12-myristate 13-acetate and then transferred to 96-well plates with white walls and flat bottoms (IWAKI, 3620-096). Using a spectral scanning multimode reader (Var-ioskan® Flash, Thermo), chemiluminescence was measured for 25 min at 37 • C with or without 2 μg/ml superoxide dismutase.

Fig. 1. p22 phox CGD mutant proteins in this study
A, amino acid sequence that corresponds to exons 3 and 5 in p22 phox . A22 • is the CGD phenotype. The "A" letter and "22" number refer to autosomal recessive and p22 phox and the superscript • indicates whether the p22 phox protein is absent based on immunoblotting [16]. Five missense mutations are located in exon 3 (L51Q, L52P, E53V, and P55R) and exon 4 (R90Q). The production of H 2 O 2 by cells expressing Nox4 was assayed using the homovanillic acid-horseradish peroxidase detection system as described previously [21,31].

Protein stabilization
Protein stability in plasmid-transfected CHO-K1 cells and HeLa cells was analyzed as described previously [21]. Briefly, the transfected cells were treated with 10 μg/ml cycloheximide (CHX) for the indicated times. When p22 phox was coexpressed with Nox2 or Nox4, the transfected cells were exposed to 10 μg/ml Brefeldin A for 1 h and then treated with 10 μg/ml cycloheximide (CHX) in the presence or absence of 20 μM MG132. The Brefeldin A treatment was performed to inhibit ER exit of the Nox-p22 phox complex to estimate the stability of the complex in the ER. To estimate the protein levels, the band intensities observed in the immunoblotting experiment were assessed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Fig. 2. Stability of p22 phox CGD mutant proteins
A, expression levels of p22 phox mutant protein. CHO-K1 (7 × 10 5 cells in 6-well plates) cells were transfected with the indicated plasmids: pcDNA3.1-wild-type (wt) ). B and C, stability of p22 phox mutant protein. CHO-K1 cells (7 × 10 5 cells in 6-well plates) were transfected with the indicated plasmids: The transfected cells were treated for 0, 1, 3, 5, or 9 h with cycloheximide (CHX) in the presence or absence of 20 μM MG132. Protein levels of the indicated proteins were estimated via immunoblotting. Positions for marker proteins are indicated in kDa. Each graph represents the relative density of the bands normalized to β-tubulin (n = 3). Statistical analysis was performed using Tukey-Kramer test. ***, p < 0.001; **, p < 0.05; *, p < 0.05; ns, no significance; shorter exposure, shorter exposure films were used for scanning; longer exposure, longer exposure films were used for scanning. These experiments have been repeated more than three times with similar results. 10 6 in a 6-cm dish) were lysed using lysis buffer (20 mM Tris-HCl, pH 7.4; 150 mM NaCl; 1% Triton X-100). For transfected CHO cells, proteins in the lysates were precipitated with ANTI-FLAG® M2 Agarose Affinity Gel or Mouse IgG-Agarose (Sigma-Aldrich). For the transfected HeLa cells, proteins in the lysates were precipitated by anti-Myc antibody or control IgG (Fig. 10A).

Cell surface biotinylation assay
Cell surface biotinylation assay was performed as described previously [31].

Statistical analysis
Data were expressed as mean ± standard deviations. Between-group comparisons were performed using t-test and Tukey-Kramer multiple comparison of means test. Statistical analyses were performed using EZR (Saitama Medical Center, Jichi Medical University, Saitama, Japan), a graphical user interface for R (The R Foundation for Statistical Computing, Vienna, Austria).
based on the fact that the L51Q, L52P, E53V, and P55R mutations lead to the A22 • type of CGD [16]. Because the amino acid substitutions may interfere with the recognition of p22 phox mutant proteins by anti-p22 phox mouse monoclonal antibody [mAB (CS9)] or rabbit polyclonal antibody, wild-type (wt) p22 phox and mutant proteins were prepared with a C-terminal Myc tag (p22 phox -Myc), which is detectable with mAB 9E10 (anti-Myc mouse monoclonal antibody).
When wild-type p22 phox -Myc was ectopically expressed alone in CHO-K1 cells, which do not express endogenous p22 phox and Nox2 [34], wild-type p22 phox -Myc was observed to be colocalized with the ER marker protein PDI under a confocal laser microscope (Fig. 1B). The mutant proteins (L51Q, L52P, E53V, and P55R) were also colocalized with PDI (Fig. 1C). The laser intensity was adjusted to capture the images for the p22 phox (L51Q) and p22 phox (L52P) mutant proteins with low expression as described in detail below (Fig. 2). Thus CHO-K1 cells expressing exogenous p22 phox proteins represent a useful system to characterize p22 phox mutant proteins in the ER.
When CHO-K1 cell lysates expressing exogenous p22 phox -Myc proteins were immunoblotted with polyclonal antibody to p22 phox and monoclonal antibody (9E10) to the Myc tag, the L51Q and L52P substitutions resulted in a decrease in the amount of the mutant protein ( Fig. 2A). This result suggests that the mutation makes the protein unstable.
We investigated the effects of amino acid substitutions on the stability of p22 phox . CHO K1 cells expressing p22 phox -Myc were treated with cycloheximide (CHX) to inhibit the de novo synthesis of p22 phox in time course experiments (0, 1, 3, 5, and 9 h). The resulting cell lysates were analyzed by immunoblotting. The levels of p22 phox (L51Q)-Myc and p22 phox (L52P)-Myc mutant proteins were decreased to approximately 40% and 20%, respectively, after exposure to CHX for 1 h (Fig. 2B). In contrast, the E53V and P55R substitutions exerted no effect on protein expression ( Fig. 2A). The levels of p22 phox (E53V)-Myc and p22 phox (P55R)-Myc mutant proteins were maintained approximately 80% and 100%, respectively, after exposure to CHX for 1 h. However, these substitutions also affected the levels of p22 phox mutant proteins after exposure to CHX for 5 h (Fig. 2B). These results suggest that these amino acid residues are responsible for the stability of p22 phox protein in the ER. The degradation of the mutant proteins was considerably suppressed in the presence of MG132 (Fig. 2C), indicating that proteasome is responsible for p22 phox degradation.
In addition to L51Q, L52P, E53V, and P55R, a missense mutation in p22 phox (R90Q) (A22 • type CGD) has not been characterized functionally in Nox2-based oxidase. A previous study suggested that this substitution impaired the interaction of p22 phox with Nox4 [35]. To determine whether p22 phox mutant proteins bind to Nox2, we expressed wild-type p22 phox -Myc or p22 phox -Myc mutant proteins together with FLAG-Nox2 (the FLAG tag was inserted at the N-terminus of Nox2). When the FLAG-Nox2 proteins were immunoprecipitated from the cell lysates of the transfected CHO-K1 cells, Nox2 did not coprecipitate p22 phox (R90Q)-Myc (Fig. 3A). The L52P and E53V mutations considerably impaired the binding of p22 phox to Nox2 to an extent same as that of the R90Q mutation. Using a cell surface biotinylation assay, we demonstrated that p22 phox (L51Q) and p22 phox (P55R), but not p22 phox (L52P) and p22 phox (E53V), localize at the plasma membrane in a Nox2 coexpression-dependent manner (Fig. 3B). In addition, complex N-glycan-bearing Nox2 (cell surface-localized form) was detected on the plasma membrane when wild-type p22 phox , p22 phox (L51Q), or p22 phox (P55R) was coexpressed. Conversely, the high-mannose form of Nox2 (ER-localized form) was only detected in the lysate coexpressing p22 phox (L52P) or p22 phox (E53V), which cannot bind to Nox2 (Fig. 3A and B). To examine whether Nox2-p22 phox complexes generate superoxide extracellularly, we expressed wild-type p22 phox -Myc or p22 phox -Myc mutant proteins together with a set of FLAG-Nox2, p67 phox , and p47 phox . Under the same expression conditions that was used for wild-type p22 phox , p22 phox (R90Q) failed to support superoxide production by Nox2, whereas the production was partially supported by the expression of p22 phox (L51Q)-Myc and p22 phox (P55R)-Myc (Fig. 3C). However, as the complex N-glycan-bearing Nox2 was not detected in the cell surface fraction coexpressing p22 phox (L52P) or p22 phox (E53V), which cannot bind to Nox2 (Fig. 3A), superoxide generation was not observed (Fig. 3C). These results suggest that a region corresponding to exon 3 is involved in the heterodimerization of p22 phox with Nox2.
(caption on next column)  2 μg), and/or pcDNA3.1-Myc-p47 phox (0.2 μg). Superoxide production was assayed using superoxide dismutase inhibitablechemiluminescence using Diogenes. Each graph represents the mean ± standard deviation of the chemiluminescence intensities integrated for 10 min after PMA stimulation from three independent transfections. C, H 2 O 2 production by Nox4. CHO-K1 cells (7 × 10 5 cells in 6-well plates) were transfected simultaneously with the indicated plasmids: pcDNA3.1-wild-type (wt) p22 phox -Myc was assayed using catalase-inhibitable fluorescence using the homovanillic acid-horseradish peroxidase detection system. Each graph represents the mean ± standard deviation of the fluorescence intensities, which were obtained from three independent transfections. Protein levels of the indicated proteins were estimated via immunoblotting. Positions for marker proteins are indicated in kDa. Statistical analysis was performed using Tukey-Kramer test. ***, p < 0.001; **, p < 0.01; *, p < 0.05; ns, no significance. These experiments have been repeated more than three times with similar results.
(caption on next page) K. Miyano et al.

Redox-sensitive cysteine residues in p22 phox
Redox-sensitive cysteine residues in Nox2 subunits p67 phox and p47 phox participate in Nox2-based oxidase [36][37][38][39]. Therefore, we focused on Cys-50, which is adjacent to the L 51 -L 52 -E 53 -P 55 sequence (Fig. 1A). We determined whether the redox-sensitive cysteine residue (s) was/were present in the ER-retained p22 phox protein. CHO-K1 cells ectopically expressing p22 phox -Myc were lysed in the presence or absence of methyl-PEG 24 -maleimide reagent [polyethylene glycol (PEG)-maleimide], which alkylates free thiol groups. PEG-maleimide-modified p22 phox was detected via immunoblotting (Fig. 4A). Following the pretreatment of cells with membrane permeable NEM, PEG-maleimide was observed to not react with the cysteines of p22 phox (Fig. 4A), suggesting that p22 phox possesses free thiol groups. When the cells are exposed to H 2 O 2 as an oxidant, redox-sensitive cysteines are expected to be oxidized. We observed that the cysteines of p22 phox were partially alkylated by PEG-maleimide because of the oxidation of the free thiol group (Fig. 4A). These results suggest that p22 phox contains redox-sensitive cysteine residues.
As shown in Fig. 1A, p22 phox contains two cysteine residues in the amino acid sequence that corresponds to exons 3 and exon 5. To determine which cysteine residue(s) are responsible for the redoxsensitivity, we expressed mutant p22 phox -Myc proteins harboring serine substitutions for Cys-50 and Cys-113 in CHO-K1 cells. As shown Fig. 4B, the C50S substitution resulted in a decrease in the amount of the mutant protein, suggesting that the mutation renders the protein unstable. In contrast, the substitution of Cys-113 exerted little effect on protein expression. The amount of p22 phox (C50S/C113S)-Myc double mutant protein was affected by the instability caused by the C50S substitution. These mutant proteins were colocalized with PDI (Fig. 4C), indicating that these mutant proteins were localized in the ER without the coexpression of Nox2. Next, the expression levels of each mutant protein were adjusted to be the same by varying the amount of plasmid used for transfection (Fig. 4D). When the transfected cells were lysed in the presence of PEG-maleimide, the apparent molecular masses of p22 phox (C50S)-Myc and p22 phox (C113S)-Myc were found to be slightly lower than that of wild-type p22 phox -Myc (Fig. 4D). The apparent molecular masses of the double mutant proteins remained unchanged in the presence or absence of PEG-maleimide (Fig. 4D). These results indicate that Cys-50 and Cys-113 are redox-sensitive.
When the transfected cells were lysed in the presence of PEGmaleimide, only PEG-maleimide-modified p22 phox (C113S)-Myc was detected via immunoblotting (Fig. 4D). This result suggests that the Cys-50 of p22 phox was readily alkylated by PEG-maleimide. Interestingly, the Cys-50 of mutant p22 phox proteins carrying the L51Q/C113S, L52P/ C113S, E53V/C113S, or P55R/C113S substitution was partially alkylated by PEG-maleimide (Fig. 4E). This result suggests that the exposure of the thiol in Cys-50, which is accessible to PEG-maleimide, might be affected by substitutions of amino acids adjacent to the Cys-50 of p22 phox .

Role of redox-sensitive Cys-50 and Cys-113 in Nox activity
Next, we investigated the role of Cys-50 and Cys-113 in the ROSgenerating activity of Nox2. Using a cell surface biotinylation assay, we demonstrated that p22 phox (C50S)-Nox2 and p22 phox (C113S)-Nox2 complexes localize at the plasma membrane (Fig. 5A). We expressed wild-type p22 phox -Myc, p22 phox (C50S)-Myc, or p22 phox (C113S)-Myc together with a set of Nox2, p67 phox , and p47 phox . Under the same expression condition that was used for wild-type p22 phox , p22 phox (P156Q)-Myc, which was defective in binding to p47 phox (a mutation found in a patient with CGD) [25,26], failed to support superoxide production by Nox2. In contrary, the production was sufficiently supported by the expression of p22 phox (C50S)-Myc and fully supported by that of p22 phox (C113S)-Myc (Fig. 5B). Nox4 also interacted with p22 phox to function as an H 2 O 2 -producing oxidase [35]. When FLAG-Nox4 and p22 phox mutant proteins were expressed, these mutant proteins activated Nox4 to the same extent as the wild-type p22 phox (Fig. 5C). These results indicate that the thiol groups of Cys-50 and Cys-113 are not required for the catalytic function of Nox.

Role of redox-sensitive Cys-50 in p22 phox protein stability
The serine substitution of redox-sensitive Cys-50, but not Cys-113, affected the protein expression level (Fig. 4B). We investigated the effect of the serine substitution of Cys-50 on the stability of p22 phox . As shown in Fig. 6A, the levels of p22 phox (C50S)-Myc mutant protein decreased to approximately 25% after exposure to CHX for 2 h. This result indicates that Cys-50 is indispensable for protein stability. The degradation of p22 phox (C50S)-Myc was considerably suppressed in the presence of MG132 (Fig. 6B), indicating that proteasome is responsible for the degradation of the p22 phox (C50S) mutant protein.
Furthermore, the oxidation of Cys-50 might affect p22 phox stabilization. To test this hypothesis, CHO-K1 cells expressing p22 phox -Myc were treated with CHX in the presence or absence of H 2 O 2 ( Fig. 6C and D), the addition of which suppressed the degradation of wild-type p22 phox -Myc and p22 phox (C113S)-Myc but not p22 phox (C50S)-Myc (Fig. 6D). The suppression effect was attenuated by pretreatment with NEM for blocking free thiols (Fig. 6C). The H 2 O 2 treatment was able to partially inhibit the proteasome-dependent degradation of wild-type p22 phox and p22 phox (C113S) mutant protein (Fig. 6E). These results indicate that redox-sensitive Cys-50 is responsible for the stability of p22 phox protein in a thiol oxidation-dependent manner.

Role of Cys-50 of p22 phox in Nox2 and Nox4 protein stability
We investigated the effect of p22 phox (C50S)-Myc on the stability of Nox2 and Nox4. The coexpression of wild-type p22 phox -Myc significantly stabilized ER-localized Nox2 (Fig. 7A) and Nox4 (Fig. 7B) carrying high-mannose glycan proteins. In contrast, these effects were not observed with p22 phox (C50S)-Myc. Nox2 and Nox4 interacted with p22 phox (C50S)-Myc as well as with wild-type p22 phox -Myc (Fig. 7C).  Myc (0.2 μg). The transfected cells were treated for 0, 2, 4, or 6 h with cycloheximide (CHX) in the presence or absence of 20 μM MG132. The graph represents the relative densities of the bands normalized to β-tubulin (n = 3). Protein levels of the indicated proteins were estimated via immunoblotting. Statistical analysis was performed using Tukey-Kramer test. ***, p < 0.001; **, p < 0.01; *, p < 0.05; ns, no significance. Positions for marker proteins are indicated in kDa. These experiments have been repeated more than three times with similar results.
(caption on next page) K. Miyano et al. Degradation of Nox2 and Nox4 was considerably suppressed in the presence of MG132 (Fig. 7D and E), indicating that proteasome is responsible for degradation of Nox2 and Nox4 complexed with p22 phox . These results indicate that the instability of p22 phox (C50S) protein affects the stability of Nox2 and Nox4 proteins when complexed with p22 phox .
In whole cell lysates, p22 phox (E53V)-Myc and p22 phox (P55R)-Myc were observed at an extent similar to that of wild-type p22 phox -Myc at a plasmid ratio of 1:1 (wild-type:mutant proteins), and Derlin-1 was strongly bound to these mutant proteins (Fig. 9A). Although the expression levels of p22 phox (L51Q)-Myc and p22 phox (L52P)-Myc were considerably lower than those of wild-type p22 phox , Derlin-1 coprecipitated these mutant proteins at an extent similar to that of wild-type p22 phox -Myc (Fig. 9A). These mutant proteins were observed to be colocalized with Derlin-1-FLAG via a confocal laser microscope (Fig. 9B). These results suggest that Derlin-1 participates in ERADmediated degradation of p22 phox . To test this possibility, we attempted to knock down Derlin-1 in HeLa cells using commercially available and validated siRNA against human Derlin-1. We observed that endogenous Derlin-1 was efficiently coprecipitated with the anti-Myc antibody but not control IgG from the cell lysates of the cells expressing exogenouse p22 phox -Myc mutant proteins (Fig. 10A). Derlin-1 knockdown partially restored the expression levels of p22 phox mutant proteins (Fig. 10B), which were markedly underexpressed compared with the wild-type protein ( Figs. 2A and 4B). Derlin-1 depletion partially suppressed the degradation of p22 phox mutant proteins (Fig. 10C). These results suggest that p22 phox mutant proteins are recognized by Derlin-1 for proteasomal degradation.

Discussion
In the present study, we demonstrated that Leu-51, Leu-52, Glu-53, and Pro-55 in the amino acid sequence that corresponds to exon 3 are responsible for p22 phox protein stability. In addition, the serine substitution of Cys-50, which is adjacent to the L 51 -L 52 -E 53 -P 55 sequence and is redox-sensitive, leads to protein instability. This instability affects the stability of Nox2 and Nox4 when complexed with p22 phox . Furthermore, blocking the free thiol of Cys-50 using alkylating agents or the serine substitution of Cys-50 promotes the association of p22 phox with Derlin-1, a key component of the ERAD system. In addition, L51Q, L52P, E53V, and P55R mutant proteins bind to Derlin-1 more efficiently than the wild-type protein. These findings suggest that the C-terminal region adjacent to Cys-50 (amino acids 50-55, including Cys-50) is responsible for p22 phox protein stability (Fig. 11).
In a previous study [40], performed screening of a library of peptides spanning the amino acid sequence of p22 phox for the inhibition of Nox2 activity. These peptides interfere with the binding of p47 phox to Nox2-p22 phox [40]: p47 phox primarily binds to a proline-rich region (residues 151-160) in the C-terminal cytosolic tail of p22 phox . Furthermore, the screening revealed that amino acid residues 47-61 are responsible for Nox2 activity [40]. These peptides may promote the dissociation of p22 phox from the Nox2-p22 phox complex. In addition, the N-terminal region of p22 phox is required for Nox2 protein maturation [41], which completely depends on binding to p22 phox . In the present study, we demonstrated that p22 phox (L52P) and p22 phox (E53V) are defective in binding to Nox2 (Fig. 3A). Therefore, residues 44-68 that correspond to exon 3 may be the region responsible for binding to Nox2.
The L52P and E53V mutations impair the binding of p22 phox to Nox2  1 μg). After fixation, the immunofluorescence signals were observed by confocal microscopy. Scale bars, 10 μm. The data are representative of results from three independent experiments. (Fig. 3). As the p22 phox protein stability depends on the complex formation with Nox2 [46], monomer p22 phox might be degraded in the phagocytes through the ERAD pathway, resulting in the A22 • type of CGD. However, L51Q and P55R mutant proteins bind Nox2, although they are unstable. It is currently unknown whether p22 phox would be degraded in phagocytes before binding to de novo Nox2 or after complex formation with Nox2. Blocking the thiol on Cys-50 by alkylation or substituting it with hydroxyl group resulted in p22 phox degradation through the ERAD pathway (Fig. 6). In contrast, the oxidation of the Cys-50 thiol group by H 2 O 2 enhanced the stability of ER-retained p22 phox protein (Fig. 6) and blocked the alkylation of thiols (Fig. 4). In addition, the degradation of the Nox2-p22 phox and Nox4-p22 phox complexes were accelerated by the serine substitution of the redox-sensitive Cys-50 (Fig. 7). Hence, the protein expression of Nox2 and Nox4 might be regulated by the modification of the Cys-50 thiol group (Fig. 11). The effects of Cys-50 modification on the stability of p22 phox appear to be important for Nox4based oxidase activity. Nox2, which is heterodimerized with p22 phox , is activated depending on complex formation with cytosolic activating proteins and Rac in response to cell stimulation. Thus, the switch for activating Nox2 is turned on or off by the formation or dissociation of the complex. In contrast, Nox4, which is heterodimerized with p22 phox , constitutively produces ROS in a cytosolic activating proteinindependent manner. Because the switch for Nox4 activity cannot be easily turned off, Nox4 degradation appears to be an effective way to turn off Nox4 activity. Nox4 protein stability is dependent on the presence of p22 phox (Fig. 7B) [22,23]. In addition, Nox4 primarily localizes in the ER [21,47,48]. Thus, we propose that the modification of the Cys-50 thiol group results in the degradation of p22 phox through the ERAD pathway and is a switch for Nox4 inactivation. Misfolded CFTR-ΔF508 membrane protein can escape ERAD through low-temperature treatment [49] or chemical (VX-809) treatment [50]. These treatments rescue CFTR-ΔF508 trafficking from the ER to the plasma membrane and partially restore the function of the chloride channel. Because p22 phox (L51Q) and p22 phox (P55R) retain the ability to associate with Nox2, the strategy of the escape of mutant proteins from the ERAD system may overcome defective Nox2-based activity in patients with A22 • type CGD. Modification of the thiol present in Cys-50, which is adjacent to the L 51 -L 52 -E 53 -P 55 sequence in the amino acid sequence that corresponds to exon 3, is responsible for both avoiding and promoting the degradation of p22 phox . Thus, the identification of molecules involved in the modification of Cys-50 thiol may be valuable for future studies. Molecules that bind/dissociate depending on modification may also be discovered. Additionally, in the present study, we used the indirect method of detecting cysteine oxidation based on reactivity loss with thiol-modifying reagents in the cell lysate. Proteomic approaches are proposed by the Chouchani [51] and Carroll [52] groups for the characterization of cysteine thiol modifications. Using their proposed methods in the future, we would identify post-translational p22phox cysteine residue modifications in intact primary phagocytes. Fig. 11. Schematic representation of the p22 phox degradation through ERAD system Regulation of Derlin-1-mediated degradation of p22 phox by thiol modification. The oxidation of the Cys-50 thiol group by H 2 O 2 enhanced the stability of ER-retained p22 phox protein (a). The thiol on Cys-50 by alkylation resulted in p22 phox degradation through the ERAD pathway (b). C50S substitution resulted in p22 phox degradation through the ERAD pathway (c).