Proteomic investigation on bio-corona of functionalized multi­walled carbon nanotubes

Background

The formation of bio-corona, due to adsorption of biomolecules onto carbon nanotubes (CNTs) surface in a physiological environment, may lead to a modified biological “identity” of CNTs, contributing to determination of their biocompatibility and toxicity.

Preparation and Toxicological Assessment of Functionalized Carbon Nanotube-Polymer Hybrids

Novel Carbon Nanotube-Polymer Hybrids were synthesized as potential materials for the development of membranes for water treatment applications in the field of Membrane Bioreactors (MBRs). Due to the toxicological concerns regarding the use of nanomaterials in water treatment as well as the rising demand for safe drinking water to protect public health, we studied the functionalization of MWCNTs and Thin-MWCNTs as to control their properties and increase their ability of embedment into porous anisotropic polymeric membranes. Following the growth of the hydrophilic monomer on the surface of the properly functionalized CNTs, that act as initiator for the controlled radical polymerization (ATRP) of sodium styrene sulfonate (SSNa), the antimicrobial quaternized phosphonium and ammonium salts were attached on CNTs-g-PSSNa through non-covalent bonding. In another approach the covalent attachment of quaternized ammonium polymeric moieties of acrylic acid-vinyl benzyl chloride copolymers with N,N-dimethylhexadecylamine (P(AA12-co-VBCHAM)) on functionalized CNTs has also been attempted. Finally, the toxicological assessment in terms of cell viability and cell morphological changes revealed that surface characteristics play a major role in the biological response of functionalized CNTs.     

Study of hepatotoxicity and oxidative stress in male Swiss-Webster mice exposed to functionalized multi-walled carbon nanotubes

Carbon nanotubes (CNTs), the most promising material with unique characteristics, find its application in different fields ranging from composite materials to medicine and from electronics to energy storage. However, little is known about the mechanisms behind the interaction of these particles with cells and their toxicity. The aim of this study was to assess the effects, after intraperitoneal (ip) injection, of functionalized multi-walled carbon nanotubes (MWCNT) (carboxyl groups) on various hepatotoxicity and oxidative stress biomarkers (ROS, LHP, ALT, AST, ALP, and morphology of liver) in the mouse model. The mice were dosed ip at 0.25, 0.5, and 0.75 mg/kg/day for 5 days of purified/functionalized MWCNTs and two controls (negative; saline and positive; carbon black 0.75 mg/kg) as appropriate. Samples were collected 24 h after the fifth day treatment following standard protocols. Exposure to carboxylated functionalized MWCNT; the body-weight gain of the mice decreased, induced reactive oxygen species (ROS), and enhanced the activities of serum amino-transferases (ALT/AST), alkaline phosphatases (ALP), and concentration of lipid hydro peroxide compared to control. Histopathology of exposed liver showed a statistically significant effect in the morphological alterations of the tissue compared to controls. The cellular findings reported here do suggest that purified carboxylated functionalized MWCNT has the potential to induce hepatotoxicity in Swiss-Webster mice through activation of the mechanisms of oxidative stress, which warrant in vivo animal exposure studies. However, more studies of functionalization in the in vivo toxicity of MWCNTs are required and parallel comparison is preferred.     

Rac1-NADPH oxidase-regulated generation of reactive oxygen species mediates glutamate-induced apoptosis in SH-SY5Y human neuroblastoma cells

Reactive oxygen species (ROS) are known to play an important role in glutamate-induced neuronal cell death. In the present study, we examined whether NADPH oxidase serves as a source of ROS production and plays a role in glutamate-induced cell death in SH-SY5Y human neuroblastoma cells. Stimulation of the cells with glutamate (100 mM) induced apoptotic cell death and increase in the level of ROS, and these effects of glutamate were significantly suppressed by the inhibitors of the NADPH oxidase, diphenylene iodonium, apocynin, and neopterine. In addition, RT-PCR revealed that SH-SY5Y cells expressed mRNA of gp91^phox, p22^phox and cytosolic p47^phox, p67^phox and p40^phox, the components of the plasma membrane NADPH oxidase. Treatment with glutamate also resulted in activation and translocation of Rac1 to the plasma membrane. Moreover, the expression of Rac1N17, a dominant negative mutant of Rac1, significantly blocked the glutamate-induced ROS generation and cell death. Collectively, these results suggest that the plasma membrane-bound NADPH oxidase complex may play an essential role in the glutamate-induced apoptotic cell death through increased production of ROS.     

Comparative pulmonary toxicity assessment of pristine and functionalized multi-walled carbon nanotubes intratracheally instilled in rats: morphohistochemical evaluations

Increasing interest in safety evaluation of carbon nanotubes (CNTs) has risen in relation to their wide applications, together with the evidence of their cytotoxic effects. It has been shown that chemical functionalization extends the applications of CNTs, conferring them new functions that cannot otherwise be acquired by pristine CNTs, but also impacts on biological response to CNTs, modifying their toxicological profile. We assessed the onset of pulmonary toxic effects caused by pristine MW-CNTs and functionalized MW-NH? or MW-COOH, 16 days after intratracheal instillation (1 mg/kg b.w.); major endpoints tested included (i) histopathology of lung (Haematoxylin/Eosin Staining), (ii) apoptotic/proliferating features examined by TUNEL and PCNA immunostaining, and (iii) presence/distribution of (1) Transforming Growth Factor-beta1 (TGF?1), (2) Interleukin-6 (IL-6) and (3) Collagen (Type I) investigated by immunochemical methods, as markers of lung toxicity, inflammation, and fibrosis, respectively. Lung histopathology from exposed animals showed dark, particulate-laden macrophages, reflecting carbon nanomaterial engulfing, both at alveolar and bronchiolar levels, after treatment with all the tested CNTs. Alteration of lung architecture was also observed in several areas showing collapsed thick-walled alveoli and the presence of micro-haemorrhagic foci. TUNEL and PCNA, indicative of apoptosis and cell proliferation respectively, showed a significant increase of immunopositive cells at bronchiolar, alveolar and macrophagic levels, as expression of an improved cellular turnover. Increased immunoreactivity for pulmonary TGF?1 and IL-6 was observed in treated rats, particularly in bronchiolar areas, collapsed alveoli and at stromal level, while evident changes for collagen were not detected. Taken together these findings demonstrated the general pulmonary toxicity coupled with inflammatory response after in vivo exposure to CNTs, without overt signs of fibrosis and granuloma formation, irrespectively of nanotube functionalization.     

NADPH oxidase subunit p22phox-mediated reactive oxygen species contribute to angiogenesis and tumor growth through AKT and ERK1/2 signaling pathways in prostate cancer

Excessive generation of reactive oxygen species (ROS) in cancer cells is associated with cancer development, but the underlying mechanisms and therapeutic significance remain elusive. In this study, we reported that levels of ROS and p22^phox expression are greatly increased in human prostate cancer tissues, and knockdown of p22^phox by specific small interfering RNA (siRNA) decreased ROS levels in prostate cancer cells. We also showed that stable downregulation of p22^phox in prostate cancer cells inhibited cell proliferation and colony formation, which was mediated by AKT and extracellular signal-regulated kinase (ERK)1/2 signaling pathways and their downstream molecules hypoxia-inducible factor 1α (HIF-1α) and vascular endothelial growth factor (VEGF). The NADPH oxidase subunit NOX1 was also elevated in prostate cancer cells, and was involved in activation of AKT/ERK/HIF-1/VEGF pathway and regulation of cell proliferation. Knockdown of p22^phox resulted in inhibition of tumor angiogenesis and tumor growth in nude mice. These findings reveal a new function of p22^phox in tumor angiogenesis and tumor growth, and suggest that p22^phox is a potential novel target for prostate cancer treatment.     

Effects of single and multi walled carbon nanotubes on macrophages: cyto and genotoxicity and electron microscopy

Production of nanotechnology-based materials is increasing worldwide: it is essential to evaluate their potential toxicity. Among these nanomaterials, carbon nanotubes (CNTs) have tremendous potential in many areas of research and applications. We have investigated the cyto- and genotoxic effects of single and multi-walled CNTs (SWCNTs, MWCNTs) and carbon black (CB) on the mouse macrophage cell line RAW 264.7. Specifically we have investigated inflammatory response, release of tumor necrosis factor-α (TNF-α), intracellular reactive oxygen species (ROS) production, cell death (both necrosis and apoptosis), chromosomal aberrations and cellular ultrastructural alteration caused by CB, MWCNTs and SWCNTs. Our data confirm that both CNTs and CB are cyto and geno-toxic to RAW 264.7 mouse macrophages. CNTs exposure induced ROS release, necrosis and chromosomal aberrations but did not cause an inflammatory response. In addition CNTs induce ultrastructural damage and apoptosis. CNTs penetrate the cell membrane and individual MWCNTs are seen associated with the nuclear envelope.     

Bilberries exert an anti-atherosclerotic effect in lipid-loaded macrophages

We hypothesized that the mechanism responsible for the anti-atherosclerotic action of bilberry extract (BE) is linked to its antioxidant and anti-inflammatory potential, and investigated its direct effect on the regulation of apolipoprotein E (apoE) and cholesteryl ester transfer protein (CETP) secretion from lipid-loaded macrophages. Human THP-1 macrophages were loaded with lipids by incubation with human copper-oxidized LDL (oxLDL) and then exposed to different concentrations of BE (1–5 µg mL^?1) obtained from bilberries (mechanically homogenized and solubilized in ethanol). Cellular and secreted proteins, the phosphorylation level of NF-κB and protein kinase A (PKA) were quantified by Western blot and gene expression was evaluated by Real-time PCR. The results showed that BE induced in lipid-loaded macrophages has: (i) an antioxidant effect by reducing the expression of NADPH oxidase subunits, p22^phox, p47^phox and NOX4, (ii) an anti-inflammatory effect by diminishing the secretion of CRP, MCP-1 and IL-1β and (iii) cholesterol efflux by increasing the secretion of apoE and CETP and by reducing cellular cholesterol content. BE exerted these effects by inhibition of NF-κB and activation of PKA signaling pathways. Our study supports BE therapeutic administration to decrease oxidative and inflammatory stress by a molecular mechanism regulated by NF-κB and PKA signaling pathways in lipid-loaded macrophages.     

Synthesis of bacterial celluloses in multiwalled carbon nanotube-dispersed medium

Carbon nanotubes are considered to be the ideal multi-functional filler, although there is some debate regarding their toxicity for bio-related applications. The bacteria, Gluconacetobacter xylinum, which produce bacterial cellulose, were cultured in a multiwalled carbon nanotube (MWCNT) dispersed Hestrin and Schramm (HS) medium by shaking incubation. The MWCNTs were functionalized with polyethylene glycol to prepare a stable MWCNT-dispersed HS medium and its stability was characterized by measuring the transmittance of a pulsed near infrared light. To investigate the toxicity of the MWCNTs to bacteria, we also introduced a green fluorescent protein gene into the bacteria and observed the fluorescence via confocal microscopy to confirm the presence of live bacteria in the MWCNT-dispersed HS medium. On the bases of the electron microscopy observations, a substantial number of MWCNTs were found to be well-dispersed and attached to the surface of the bacterial cellulose fibrils.     

Facile surface functionalization of multiwalled carbon nanotubes by soft dielectric barrier discharge plasma: Generate compatible interface for lipase immobilization

To create compatible interface for enzyme immobilization, the surface of multi-walled carbon nanotubes (MWCNTs) was functionalized using soft technique dielectric barrier discharge plasma (DBDP) for carboxylation and amination; followed by further amidation of carboxyl group with alkylamine. Successful functionalization and enzyme immobilization were structurally confirmed using spectroscopic analysis Fourier-Transform Infrared Spectroscopy (FTIR) and X-ray Photoelectron Spectroscopy (XPS). The immobilization of Candida rugosa lipase (CRL) on functionalized MWCNTs was evidenced by clearly viewing with Transmission Electron Microscopy (TEM) and Atomic Force Microscopy (AFM) imaging. CRL showed more Freundlich equilibrium behavior upon immobilization on annealed and octadecylamidated MWCNTs, which suggested a multilayer adsorption; while upon physical adsorption on aminated and carboxylated MWCNTs, CRL, to more extent, demonstrated a Langmuir equilibrium property, producing an enzyme monolayer. It was proven that DBDP-mediated surface-functionalization could create compatible microenvironments for enzyme immobilization, resulted in improved specific activity and thermostability. The immobilized CRL on octadecylamidated MWCNTs displayed excellent reusability and operation stability, indicating its potential for industrial application.     

Aryl hydrocarbon receptor-dependent induction of the NADPH oxidase subunit NCF1/p47 expression leading to priming of human macrophage oxidative burst

Polycyclic aromatic hydrocarbons such as benzo(a)pyrene (BaP) are toxic environmental contaminants known to regulate gene expression through activation of the aryl hydrocarbon receptor (AhR). In the present study, we demonstrated that acute treatment by BaP markedly increased expression of the NADPH oxidase subunit gene neutrophil cytosolic factor 1 (NCF1)/p47^phox in primary human macrophages; NCF1 was similarly up-regulated in alveolar macrophages from BaP-instilled rats. NCF1 induction in BaP-treated human macrophages was prevented by targeting AhR, through its chemical inhibition or small interference RNA-mediated down-modulation of its expression. BaP moreover induced activity of the NCF1 promoter sequence, containing a consensus AhR-related xenobiotic-responsive element (XRE), and electrophoretic mobility shift assays and chromatin immunoprecipitation experiments indicated that BaP-triggered binding of AhR to this XRE. Finally, we showed that BaP exposure resulted in p47^phox protein translocation to the plasma membrane and in potentiation of phorbol myristate acetate (PMA)-induced superoxide anion production in macrophages. This BaP priming effect toward NADPH oxidase activity was inhibited by the NADPH oxidase specific inhibitor apocynin and the chemical AhR inhibitor α-naphtoflavone. These results indicated that BaP induced NCF1/p47^phox expression and subsequently enhanced superoxide anion production in PMA-treated human macrophages, in an AhR-dependent manner; such an NCF1/NADPH oxidase regulation by polycyclic aromatic hydrocarbons may participate in deleterious effects toward human health triggered by these environmental contaminants, including atherosclerosis and smoking-related diseases.     

Biosafety of non-surface modified carbon nanocapsules as a potential alternative to carbon nanotubes for drug delivery purposes

Background

Carbon nanotubes (CNTs) have found wide success in circuitry, photovoltaics, and other applications. In contrast, several hurdles exist in using CNTs towards applications in drug delivery. Raw, non-modified CNTs are widely known for their toxicity. As such, many have attempted to reduce CNT toxicity for intravenous drug delivery purposes by post-process surface modification. Alternatively, a novel sphere-like carbon nanocapsule (CNC) developed by the arc-discharge method holds similar electric and thermal conductivities, as well as high strength. This study investigated the systemic toxicity and biocompatibility of different non-surface modified carbon nanomaterials in mice, including multi-walled carbon nanotubes (MWCNTs), single-walled carbon nanotubes (SWCNTs), carbon nanocapsules (CNCs), and C₆₀ fullerene (C₆₀). The retention of the nanomaterials and systemic effects after intravenous injections were studied.

Methodology and Principal Findings

MWCNTs, SWCNTs, CNCs, and C₆₀ were injected intravenously into FVB mice and then sacrificed for tissue section examination. Inflammatory cytokine levels were evaluated with ELISA. Mice receiving injection of MWCNTs or SWCNTs at 50 µg/g b.w. died while C₆₀ injected group survived at a 50% rate. Surprisingly, mortality rate of mice injected with CNCs was only at 10%. Tissue sections revealed that most carbon nanomaterials retained in the lung. Furthermore, serum and lung-tissue cytokine levels did not reveal any inflammatory response compared to those in mice receiving normal saline injection.

Conclusion

Carbon nanocapsules are more biocompatible than other carbon nanomaterials and are more suitable for intravenous drug delivery. These results indicate potential biomedical use of non-surface modified carbon allotrope. Additionally, functionalization of the carbon nanocapsules could further enhance dispersion and biocompatibility for intravenous injection.     

NADPH-oxidase activation and cognition in Alzheimer disease progression

Superoxide production via NADPH-oxidase (NOX) has been shown to play a role in a variety of neurological disorders, including Alzheimer disease (AD). To improve our understanding of the NOX system and cognitive impairment, we studied the various protein components of the phagocytic isoform (gp91^phox, or NOX2) in the frontal and temporal cortex of age- and postmortem-matched samples. Individuals underwent antemortem cognitive testing and postmortem histopathologic assessment to determine disease progression and assignment to one of the following groups: no cognitive impairment (NCI), preclinical AD, mild cognitive impairment (MCI), early AD, and mild-to-moderate AD. Biochemical methods were used to determine overall NOX activity as well as levels of the various subunits (gp91^phox, p67^phox, p47^phox, p40^phox, and p22^phox). Overall enzyme activity was significantly elevated in the MCI cohort in both cortical regions compared to the NCI cohort. This activity level remained elevated in the AD groups. Only the NOX cytosolic subunit proteins (p67^phox, p47^phox, and p40^phox ) were significantly elevated with disease progression; the membrane-bound subunits (gp91^phox and p22^phox) remained stable. In addition, there was a robust correlation between NOX activity and the individual's cognitive status such that as the enzyme activity increased, cognitive performance decreased. Collectively, these data show that upregulated NADPH-oxidase in frontal and temporal cortex suggests that increases in NOX-associated redox pathways might participate in early pathogenesis and contribute to AD progression.     

Immobilization and In vitro Evaluation of Soyasapogenol B onto Functionalized Multi-Walled Carbon Nanotubes

Context: Soyasapogenol B (SSB) has been shown to possess hepatoprotective, antiviral, anti-inflammatory, antimutagenic and anticancer activities. The goal of this work is to study the influence of functionalized multi-walled carbon nanotubes (MWCNTs) on the biological activity of the loaded soyasapogenol B.Methods: SSB was prepared by enzymatic hydrolysis of soybean saponin using Aspergillus flavus whole cells. While, the functionalization of MWCNTs was conducted using the adsorption technique in the presence of the modified montmorillonite (mMMT) with cetyltrimethyl ammonium bromide (CTAB). In vitro drug release profile, kinetics of release, cellular uptake and cytotoxicity were also investigated. The prepared materials were characterized using: FTIR, particle size distribution analysis and TEM.Results: The in vitro release and cytotoxicity of the SSB loaded and unloaded samples were carried out using the dialysis bag diffusion technique and sulphorhodamine B (SRB) assay, respectively. The results showed that SSB loaded MWCNTs, mMMT and MWCNTs/mMMT had particle size of 414, 1121 and 412 nm, respectively, and 338, 1071 and 268 nm, for the unloaded ones, respectively. FTIR proved that SSB was successful immobilized onto functionalized MWCNTs.Conclusions: Successful loading of SSB, as a bioactive material, onto functionalized MWCNTs with modified clay (montmorillonite) using CTAB exhibited very low cytotoxic behavior with human normal melanocyte (HFB-4), breast (MCF-7) and liver (Hep-G2) carcinoma cell lines relative to SSB and doxorubicin standard.     

The impact of multi-walled carbon nanotubes (MWCNTs) on macrophages: contribution of MWCNT characteristics

Multi-walled carbon nanotubes(MWCNTs) have wide application prospects but also exhibit notable biotoxicity that is tightly associated with macrophages. Macrophages simultaneously act as initiators and defenders in MWCNT-induced organ lesions,and targeting macrophages with MWCNTs may be a potential immunotherapy and oncotherapy approach. This review focuses on the impacts of MWCNTs on macrophages and further discusses the influence of MWCNT characteristics on their bioactivity.Based on existing studies, MWCNTs stimulate macrophage migration, induce secretion of various cytokines and activate inflammatory pathways in macrophages, especially NLRP3-mediated IL-1β production. This inflammatory state, together with the oxidative stress and cell membrane lesions induced by MWCNTs, contributes to decreased phagocytic ability and cell viability, which finally results in cell apoptosis and necrosis. A series of intracellular and systemic components, such as toll-like receptor, high-mobility group box 1, Rho-associated kinases, scavenger receptor and complement components, may be involved in the above-mentioned cell-MWCNT interactions. The characteristics of MWCNTs can influence their bioactivity in macrophages both mechanically and chemically. The size(length and/or diameter), functionalization, purification and even the experimental method can affect the influence of MWCNTs on macrophages, and a better understanding of these MWCNT characteristics may benefit utilization of this nanomaterial in associated nanomedical applications.     

A Conserved Region between the TPR and Activation Domains of p67phox Participates in Activation of the Phagocyte NADPH Oxidase

The phagocyte NADPH oxidase, dormant in resting cells, is activated during phagocytosis to produce superoxide, a precursor of microbicidal oxidants. The membrane-integrated protein gp91^phox serves as the catalytic core, because it contains a complete electron-transporting apparatus from NADPH to molecular oxygen for superoxide production. Activation of gp91^phox requires the cytosolic proteins p67^phox, p47^phox, and Rac (a small GTPase). p67^phox, comprising 526 amino acids, moves upon cell stimulation to the membrane together with p47^phox and there interacts with Rac; these processes are prerequisite for gp91^phox activation. Here we show that a region of p67^phox (amino acids 190–200) C-terminal to the Rac-binding domain is evolutionarily well conserved and participates in oxidase activation at a later stage in conjunction with an activation domain. Alanine substitution for Tyr-198, Leu-199, or Val-204 abrogates the ability of p67^phox to support superoxide production by gp91^phox-based oxidase as well as its related oxidases Nox1 and Nox3; the activation also involves other invariant residues such as Leu-193, Asp-197, and Gly-200. Intriguingly, replacement of Gln-192 by alanine or that of Tyr-198 by phenylalanine or tryptophan rather enhances superoxide production by gp91^phox-based oxidase, suggesting a tuning role for these residues. Furthermore, the Y198A/V204A or L199A/V204A substitution leads to not only a complete loss of the activity of the reconstituted oxidase system but also a significant decrease in p67^phox interaction with the gp91^phox NADPH-binding domain, although these mutations affect neither the protein integrity nor the Rac binding activity. Thus the extended activation domain of p67^phox (amino acids 190–210) containing the D(Y/F)LGK motif plays an essential role in oxidase activation probably by interacting with gp91^phox.     

Nitroarachidonic acid prevents NADPH oxidase assembly and superoxide radical production in activated macrophages

Nitration of arachidonic acid (AA) to nitroarachidonic acid (AANO₂) leads to anti-inflammatory intracellular activities during macrophage activation. However, less is known about the capacity of AANO₂ to regulate the production of reactive oxygen species under proinflammatory conditions. One of the immediate responses upon macrophage activation involves the production of superoxide radical (O₂^•−) due to the NADPH-dependent univalent reduction of oxygen to O₂^•− by the phagocytic NADPH oxidase isoform (NOX2), the activity of NOX2 being the main source of O₂^•− in monocytes/macrophages. Because the NOX2 and AA pathways are connected, we propose that AANO₂ can modulate macrophage activation by inhibiting O₂^•− formation by NOX2. When macrophages were activated in the presence of AANO₂, a significant inhibition of NOX2 activity was observed as evaluated by cytochrome c reduction, luminol chemiluminescence, Amplex red fluorescence, and flow cytometry; this process also occurs under physiological mimic conditions within the phagosomes. AANO₂ decreased O₂^•− production in a dose- (IC₅₀=4.1±1.8 μM AANO₂) and time-dependent manner. The observed inhibition was not due to a decreased phosphorylation of the cytosolic subunits (e.g., p40^phox and p47^phox), as analyzed by immunoprecipitation and Western blot. However, a reduction in the migration to the membrane of p47^phox was obtained, suggesting that the protective actions involve the prevention of the correct assembly of the active enzyme in the membrane. Finally, the observed in vitro effects were confirmed in an in vivo inflammatory model, in which subcutaneous injection of AANO₂ was able to decrease NOX2 activity in macrophages from thioglycolate-treated mice.     

ROS and NF-κB are involved in upregulation of IL-8 in A549 cells exposed to multi-walled carbon nanotubes

Carbon nanotubes (CNTs) have potential applications in biosensors, tissue engineering, and biomedical devices because of their unique physico-chemical, electronic and mechanical properties. However, there is limited literature data available concerning the biological properties and toxicity of CNTs. This study aimed to assess the toxicity exhibited by multi-walled CNTs (MWCNTs) and to elucidate possible molecular mechanisms underlying the biological effects of MWCNTs in A549 cells. Exposing A549 cells to MWCNTs led to cell death, changes in cell size and complexity, reactive oxygen species (ROS) production, interleukin-8 (IL-8) gene expression and nuclear factor (NF)-κB activation. Treatment of A549 cells with antioxidants prior to adding MWCNTs decreased ROS production and abrogated expression of IL-8 mRNA. Pretreatment of A549 cells with NF-κB inhibitors suppressed MWCNTs-induced IL-8 mRNA expression. These results indicate that MWCNTs are able to induce expression of IL-8 in A549 cells, at least in part, mediated by oxidative stress and NF-κB activation.     

Identification of NoxD/Pro41 as the homologue of the p22phox NADPH oxidase subunit in fungi

NADPH oxidases (Nox) are membrane complexes that produce O₂^?. Researches in mammals, plants and fungi highlight the involvement of Nox‐generated ROS in cell proliferation, differentiation and defense. In mammals, the core enzyme gp91^phox/Nox2 is associated with p22^phox forming the flavocytochrome b₅₅₈ ready for activation by a cytosolic complex. Intriguingly, no homologue of the p22^phox gene has been found in fungal genomes, questioning how the flavoenzyme forms. Using whole genome sequencing combined with phylogenetic analysis and structural studies, we identify the fungal p22^phox homologue as being mutated in the Podospora anserina mutant IDC⁵⁰⁹. Functional studies show that the fungal p22^phox, PaNoxD, acts along PaNox1, but not PaNox2, a second fungal gp91^phox homologue. Finally, cytological analysis of functional tagged versions of PaNox1, PaNoxD and PaNoxR shows clear co‐localization of PaNoxD and PaNox1 and unravel a dynamic assembly of the complex in the endoplasmic reticulum and in the vacuolar system.