bims-noxint Biomed News
on NADPH oxidases in tumorigenesis
Issue of 2019–06–09
27 papers selected by
Laia Caja Puigsubira, Uppsala University
  1. Mammalian NADPH Oxidases.
  2. Estradiol and NADPH oxidase crosstalk regulates responses to high fat feeding in female mice.
  3. Methods for Detection of NOX-Derived Superoxide Radical Anion and Hydrogen Peroxide in Cells.
  4. Model Systems to Investigate NOX-Dependent Cell Migration and Invasiveness.
  5. Anagliptin protects neuronal cells against endogenous amyloid β (Aβ)-induced cytotoxicity and apoptosis.
  6. Isolation of Redox-Active Endosomes (Redoxosomes) and Assessment of NOX Activity.
  7. Imaging Intestinal ROS in Homeostatic Conditions Using L-012.
  8. Proteomic Methods to Evaluate NOX-Mediated Redox Signaling.
  9. The Molecular Regulation and Functional Roles of NOX5.
  10. Hydro-Cy3-Mediated Detection of Reactive Oxygen Species In Vitro and In Vivo.
  11. Using Synthetic Peptides for Exploring Protein-Protein Interactions in the Assembly of the NADPH Oxidase Complex.
  12. Visualization of Intracellular Hydrogen Peroxide with the Genetically Encoded Fluorescent Probe HyPer in NIH-3T3 Cells.
  13. Gastrointestinal Complications in Chronic Granulomatous Disease.
  14. Protein-Protein Interaction Assay to Analyze NOX4/p22phox Heterodimerization.
  15. NADPH Oxidases and Aging Models of Lung Fibrosis.
  16. Phosphorylation of gp91phox/NOX2 in Human Neutrophils.
  17. Soluble Regulatory Proteins for Activation of NOX Family NADPH Oxidases.
  18. Guidelines for the Detection of NADPH Oxidases by Immunoblot and RT-qPCR.
  19. Intersecting Stories of the Phagocyte NADPH Oxidase and Chronic Granulomatous Disease.
  20. The X-CGD PLB-985 Cell Model for NOX2 Structure-Function Analysis.
  21. Purification and Characterization of DUOX Peroxidase Homology Domains (PHDs).
  22. NOX5 Cell-Free Assay for the High-Throughput Screening of Small Molecules.
  23. High-Throughput Screening of NOX Inhibitors.
  24. Insights into the NOX NADPH Oxidases Using Heterologous Whole Cell Assays.
  25. Rational Design and Delivery of NOX-Inhibitory Peptides.
  26. Spectroscopy of NOX Protein Family Members.
  27. Functional Characterization of DUOX Enzymes in Reconstituted Cell Models.
  1. Methods Mol Biol. 2019 ;1982 17-36
    Mammalian NADPH Oxidases.
    Hélène Buvelot, Vincent Jaquet, Karl-Heinz Krause.
      Reactive oxygen species (ROS) are highly reactive oxygen derivatives. Initially, they were considered as metabolic by-products (of mitochondria in particular), which consistently lead to aging and disease. Over the last decades, however, it became increasingly apparent that virtually all eukaryotic cells possess specifically ROS-producing enzymes, namely, NOX NADPH oxidases. In most mammals, there are seven NOX isoforms: three closely related isoforms, NOX1, 2, 3, which are activated by cytoplasmic subunits; NOX4, which appears to be constitutively active; and the EF-hand-containing Ca2+-activated isoforms NOX5 and DUOX1 and 2. Loss-of-function mutations in NOX genes can lead to serious human disease. NOX2 deficiency leads to primary immune deficiency, while DUOX2 deficiency presents as congenital hypothyroidism. Nox-deficient mice provide important tools to explore the physiological functions of various NADPH oxidases as a loss of function in Nox2, Nox3, and Duox2 leads to a spontaneous phenotype. The genetic absence of Nox1, Nox4, and Duox1 does not result in an obvious mouse phenotype (the NOX5 gene is absent in rodents and can therefore not be studied using knockout mice). Since the discovery of the NOX family at the turn of the millennium, much progress in understanding the biochemistry and the physiology of NOX has been made; however many questions remain unanswered to date. This chapter is an overview of our present knowledge on mammalian NOX/DUOX enzymes.
    Keywords:  Genetic deficiency; Mouse models; NADPH oxidase; Reactive oxygen species; Redox signaling
    DOI:  https://doi.org/10.1007/978-1-4939-9424-3_2
  2. Exp Biol Med (Maywood). 2019 Jun 04. 1535370219853563
    Estradiol and NADPH oxidase crosstalk regulates responses to high fat feeding in female mice.
    Martin J Ronis, Michael L Blackburn, Kartik Shankar, Matthew Ferguson, Mario A Cleves, Thomas M Badger.
       IMPACT STATEMENT: Estrogens are known to regulate body composition. In addition, reactive oxygen species (ROS) produced by the action of NADPH oxidase (NOX) enzymes have been linked to obesity development. We examined development of obesity and adipose tissue injury in response to feeding "Western" diets high in fat and cholesterol in intact, ovariectomized (OVX), and estrogen-replaced (OVX + E2) wild type and p47phox-/- female mice where NOX2 activity is inhibited. Weight gain, gonadal fat pad weight, and adipose tissue inflammation were greater in intact WT vs. p47phox-/- mice. Genotype effects on body weight/fat mass were abolished after OVX and restored in OVX + E2 mice. These data indicate adipose tissue responses to feeding the "Western" diet is regulated by negative cross-talk between NOX-dependent ROS signaling and E2-signaling during development. Loss of estrogens post menopause may increase the risk of obesity and metabolic syndrome as the result disinhibition of ROS signaling.
    Keywords:  Estradiol; NADPH-oxidase; adiposity obesity
    DOI:  https://doi.org/10.1177/1535370219853563
  3. Methods Mol Biol. 2019 ;1982 233-241
    Methods for Detection of NOX-Derived Superoxide Radical Anion and Hydrogen Peroxide in Cells.
    Fiona Augsburger, Aleksandra Filippova, Vincent Jaquet.
      NADPH oxidases (NOX) are transmembrane enzymes, which catalyze the formation of reactive oxygen species (ROS). In humans and most mammals, the NOX family comprises seven members, namely, NOX1-5 and the dual oxidases DUOX1 and 2. The primary product of most NOX isoforms is the superoxide radical anion O2 ċ-, which is rapidly dismutated in hydrogen peroxide (H2O2), while NOX4 and DUOX mostly generate H2O2. ROS are multifunctional molecules in tissues, and NOX-derived ROS cellular functions are as diverse as microbial killing (NOX2), thyroid hormone synthesis (DUOX2), or otoconia formation in the inner ear (NOX3). NOX are potential pharmacological targets in numerous diseases such as diabetes, fibrosis, and brain ischemia, and NOX inhibitors are currently under development. Here we describe two cellular assays to detect extracellular O2 ċ- and H2O2 in cells overexpressing specific NOX isoforms and their subunits.
    Keywords:  Amplex red; Cellular assay; Hydrogen peroxide; NADPH oxidase; Peroxidase; Superoxide radical anion; WST-1
    DOI:  https://doi.org/10.1007/978-1-4939-9424-3_13
  4. Methods Mol Biol. 2019 ;1982 473-485
    Model Systems to Investigate NOX-Dependent Cell Migration and Invasiveness.
    Howard E Boudreau, Thomas L Leto.
      There is mounting evidence indicating that reactive oxygen species (ROS) play a crucial role in cell migration and invasion. Our previous studies have demonstrated the NADPH oxidase (NOX) family of enzymes are a source of ROS in different cell types undergoing migration. Several NOX enzymes are induced or activated in processes including wound repair and maintenance of epithelial barriers, as well as in promoting metastatic cell migration and invasiveness. This chapter outlines three different in vitro assays used to examine how NOX enzymes are involved in cell motility: scratch-wound repair, Matrigel invasion, and migration from confluent cell monolayer boundaries created by cell culture inserts. The three methods provide a range of experimental approaches for delineating roles of NOX enzymes in cell migration through manipulation of the expression or activities of the endogenous or overexpressed oxidases.
    Keywords:  Cell invasion; Cell migration; NADPH oxidase; NOX1; NOX4; TGF-β; Wound healing; p53
    DOI:  https://doi.org/10.1007/978-1-4939-9424-3_28
  5. Artif Cells Nanomed Biotechnol. 2019 Dec;47(1): 2213-2220
    Anagliptin protects neuronal cells against endogenous amyloid β (Aβ)-induced cytotoxicity and apoptosis.
    Zhenbo Chen, Shanwei Tao, Xiaohui Li, Xudong Zeng, Mirong Zhang, Qinghe Yao.
      Excessive generation and accumulation of amyloid-β (Aβ) fragments by familial mutations of amyloid precursor protein (APP) and presenilin 1 (PS1) play a key role in causing oxidative stress, mitochondrial abnormalities and neuronal apoptosis in Alzheimer's disease (AD). Anagliptin, a novel DPP-4 inhibitor, is a clinical drug for the management of type II diabetes approved for use in 2012. Little on the pharmacological function of anagliptin against Aβ-induced cytotoxicity in neuronal cells is known. Here, we examined the protective capacities of anagliptin against cytotoxicity in N2a neuronal cells overexpressing APP Swedish mutant and PS1 exon 9 deletion mutant (N2a/Swe.D9). Our results demonstrate that anagliptin reduced the production of ROS and the expression of NADPH oxidase 4 (NOX-4) in N2a/Swe.D9 cells. We also reported that anagliptin activates the antioxidant system by increasing the level of reduced glutathione (GSH) and glutathione peroxidase (GPx) activity. Notably, anagliptin is able to improve mitochondrial function by elevating mitochondrial membrane potential (MMP) and adenosine triphosphate (ATP) production. Additionally, our results demonstrate that anagliptin decreased the vulnerability of cells to hydrogen peroxide (H2O2)-induced secondary insult by increasing cell viability and reducing the secretion of lactate dehydrogenase (LDH) and high mobility group box 1 protein (HMGB-1). Importantly, we found that treatment with anagliptin suppressed the mitochondrial-dependent apoptosis pathway by preventing the translocation of cytochrome C, reducing cleavage of caspase-3, and the inhibiting expression of Bax. These results implicate that anagliptin may have potential as a therapeutic agent for AD treatment.
    Keywords:  Alzheimer’s disease; amyloid-β; apoptosis; mitochondria; reactive oxygen species (ROS)
    DOI:  https://doi.org/10.1080/21691401.2019.1609979
  6. Methods Mol Biol. 2019 ;1982 461-472
    Isolation of Redox-Active Endosomes (Redoxosomes) and Assessment of NOX Activity.
    Weam S Shahin, John F Engelhardt.
      Reactive oxygen species (ROS) convey signals essential for proliferation, maintenance, and senescence of a growing list of cell types. Compartmentalization of these signals is integral to cell viability as well as the signaling pathways ROS direct. Redox-active endosomes (redoxosomes) are formed downstream of several ligand-activated receptors. NADPH oxidase (NOX) is a main component of redoxosomes, which recruits multiple proteins (Rac1, NOX2, p67phox, SOD1). Isolation of redoxosomes and evaluation of how superoxide (O2˙-) production directs receptor signaling at the level of the endosome have enabled a better understanding of biologic processes controlled by ROS. In this chapter, we will first review the major signaling pathways that utilize redoxosomes and components that control its redox-dependent functions. We will then outline biochemical and biophysical methods for the isolation and characterization of redoxosome properties.
    Keywords:  EPR; Immuno-affinity isolation; Iodixanol; Lucigenin; NOX; ROS; Rac1; Redoxosomes; TRAF
    DOI:  https://doi.org/10.1007/978-1-4939-9424-3_27
  7. Methods Mol Biol. 2019 ;1982 313-327
    Imaging Intestinal ROS in Homeostatic Conditions Using L-012.
    Emer Conroy, Gabriella Aviello.
      Reactive oxygen species (ROS) are critical redox regulators of cellular dynamics controlling homeostasis. Although numerous fluorescent probes are currently available to measure ROS in cell-based assays, the short-lived nature of these molecules renders their detection challenging in more complex biological systems, such as the gastrointestinal tract in vivo. However, in the past decade, significant progress has been made in the development of novel imaging technologies and probes, facilitating ROS quantification with high sensitivity, selectivity, and temporal resolution. The IVIS Spectrum (PerkinElmer) is an optical imaging system for small animal imaging allowing precise and noninvasive visualization of fluorescent or bioluminescent signals. Here, we describe a reproducible and comprehensive method for the measurement of physiological intestinal NADPH oxidase-derived ROS by using the chemiluminescent probe L-012. Using transgenic mice deficient in Nox isoforms expressed in the intestinal mucosa, we delineate the contribution of gut epithelial versus immune cell NADPH oxidase activity in homeostatic conditions. We also discuss L-012 probe specificity and potential alternatives for in vivo studies.
    Keywords:  IVIS; Inflammatory bowel disease; Intestine; L-012; Microbiota; NADPH oxidase; NOX1; NOX2; p22phox
    DOI:  https://doi.org/10.1007/978-1-4939-9424-3_19
  8. Methods Mol Biol. 2019 ;1982 497-515
    Proteomic Methods to Evaluate NOX-Mediated Redox Signaling.
    Christopher M Dustin, Milena Hristova, Caspar Schiffers, Albert van der Vliet.
      The NADPH oxidase (NOX) family of proteins is involved in regulating many diverse cellular processes, which is largely mediated by NOX-mediated reversible oxidation of target proteins in a process known as redox signaling. Protein cysteine residues are the most prominent targets in redox signaling, and to understand the mechanisms by which NOX affect cellular pathways, specific methodology is required to detect specific oxidative cysteine modifications and to identify targeted proteins. Among the many potential redox modifications involving cysteine residues, reversible modifications most relevant to NOX are sulfenylation (P-SOH) and S-glutathionylation (P-SSG), as both can induce structural or functional alterations. Various experimental approaches have been developed to detect these specific modifications, and this chapter will detail state-of-the-art methodology to selectively evaluate these modifications in specific target proteins in relation to NOX activation. We also discuss some of the limitations of these procedures and potential complementary approaches.
    Keywords:  DUOX; Dimedone; H2O2; NADPH oxidases; Redox signaling; S-glutathionylation; Sulfenylation
    DOI:  https://doi.org/10.1007/978-1-4939-9424-3_30
  9. Methods Mol Biol. 2019 ;1982 353-375
    The Molecular Regulation and Functional Roles of NOX5.
    David J R Fulton.
      NOX (NADPH oxidases) are a family of NADPH-dependent transmembrane enzymes that synthesize superoxide and other reactive oxygen species. There are seven isoforms (NOX1-5 and DUOX1-2) which derive from a common ancestral NOX. NOX enzymes are distinguished by different modes of activation, the types of ROS that are produced, the cell types where they are expressed, and distinct functional roles. NOX5 was one of the earliest eukaryotic Nox enzymes to evolve and ironically the last isoform to be discovered in humans. In the time since its discovery, our knowledge of the regulation of NOX5 has expanded tremendously, and we now have a more comprehensive understanding of the molecular mechanisms underlying NOX5-dependent ROS production. In contrast, the cell types where NOX5 is robustly expressed and its functional significance in health and disease remain an underdeveloped area. The goal of this chapter is to provide an up-to-date overview of the mechanisms regulating NOX5 function and its importance in human physiology and pathophysiology.
    Keywords:  Function; NADPH oxidase 5; NOX5; Posttranslational regulation; Reactive oxygen species
    DOI:  https://doi.org/10.1007/978-1-4939-9424-3_22
  10. Methods Mol Biol. 2019 ;1982 329-337
    Hydro-Cy3-Mediated Detection of Reactive Oxygen Species In Vitro and In Vivo.
    Bejan J Saeedi, Bindu Chandrasekharan, Andrew S Neish.
      Reactive oxygen species (ROS) are potent signaling molecules with critical roles in cellular pathology and homeostasis. They are produced in all cell types via a diverse array of cellular machinery, giving rise to an equally diverse repertoire of molecular effects. These range from cytotoxic killing of microbes to alteration of the cellular transcriptional response to stress. Despite their importance, research into ROS has been difficult given their inherent instability and transient signaling properties. Herein we describe methods for the use of the redox-sensitive probe hydro-Cy3 for the detection and quantification of ROS both in vitro and in vivo.
    Keywords:  Hydro-Cy3; NADPH oxidase; NOX; Oxidative stress; ROS; Reactive oxygen species
    DOI:  https://doi.org/10.1007/978-1-4939-9424-3_20
  11. Methods Mol Biol. 2019 ;1982 377-415
    Using Synthetic Peptides for Exploring Protein-Protein Interactions in the Assembly of the NADPH Oxidase Complex.
    Edgar Pick.
      The NADPH oxidase complex, responsible for reactive oxygen species (ROS) generation by phagocytes, consists of a membrane-associated flavocytochrome b 558 (a heterodimer of NOX2 and p22phox) and the cytosolic components p47phox, p67phox, Rac(1 or 2), and p40phox. NOX2 carries all redox stations through which electrons flow from NADPH to molecular oxygen, to generate the primary ROS, superoxide. For the electron flow to start, a conformational change in NOX2 is required. The dominant hypothesis is that this change is the result of the interaction of NOX2 with one or more of the cytosolic components (NADPH oxidase assembly). At the most basic level, assembly is the sum of several protein-protein interactions among oxidase components. This chapter describes a reductionist approach to the identification of regions in oxidase components involved in assembly. This approach consists of "transforming" one component in an array of overlapping synthetic peptides and assessing binding to the peptides of another component, represented by a recombinant protein. The peptides are tagged with biotin, at the N- or C-terminus, and immobilized on streptavidin-coated 96-well plates. The protein partners are expressed with a 6His tag and added to the plates in the fluid phase. Binding of the protein to the peptides is quantified by a kinetic ELISA , using a peroxidase-conjugated anti-polyhistidine antibody. Protein-peptide binding assays were applied successfully to (a) identifying the binding site on one component (represented by peptides) for another component (proteins), (b) precisely defining the "binding sequence," (c) acquiring information on the binding site in the partner protein, (d) investigating the effect of conformational changes in proteins on binding to peptides, (e) determining the effect of physicochemical modification of peptides on binding of proteins, and (f) identifying epitopes recognized by anti-oxidase component antibodies by binding of antibody to peptide arrays derived from the component.
    Keywords:  Cytochrome b 558; Kinetic ELISA; NADPH oxidase; NOX2; Peptide walking; Protein-peptide binding; Protein-protein interaction; Superoxide; Synthetic peptides; p67phox
    DOI:  https://doi.org/10.1007/978-1-4939-9424-3_23
  12. Methods Mol Biol. 2019 ;1982 259-274
    Visualization of Intracellular Hydrogen Peroxide with the Genetically Encoded Fluorescent Probe HyPer in NIH-3T3 Cells.
    Yulia G Ermakova, Nataliya M Mishina, Carsten Schultz, Vsevolod V Belousov.
      Reactive oxygen species (ROS) are involved in regulating normal physiological cell functions as second messengers as well as nonspecific damage of biomolecules in a pathological process known as oxidative stress. The HyPer family of genetically encoded probes are a useful noninvasive tool for monitoring the real-time dynamics of ROS in individual cells or model organisms. HyPer, the first genetically encoded probe for detection of hydrogen peroxide (H2O2), is oxidized with high specificity and sensitivity by H2O2, leading to ratiometric changes in the fluorescence excitation spectrum of the probe. These changes can be detected with a wide range of commercial confocal and wide-field microscope systems. Here we describe a detailed protocol for ratiometric monitoring of H2O2 produced by D-amino acid oxidase (DAAO) or by NADPH oxidase (NOX) in NIH-3T3 cells using the HyPer probe.
    Keywords:  D-amino acid oxidase; DAAO; HyPer; HyPerRed; Hydrogen peroxide; Microscopy; PDGF; ROS; Ratiometric imaging
    DOI:  https://doi.org/10.1007/978-1-4939-9424-3_15
  13. Methods Mol Biol. 2019 ;1982 573-586
    Gastrointestinal Complications in Chronic Granulomatous Disease.
    E Liana Falcone, Steven M Holland.
      Almost half of patients with chronic granulomatous disease (CGD) suffer from gastrointestinal (GI) inflammation, the pathogenesis of which is complex and multifactorial. As a result, the management of CGD-associated GI inflammation remains challenging due to its chronicity and difficulty in managing the simultaneous need for immunomodulation with increased susceptibility to infection. In order to contextualize prospective treatment interventions for CGD-associated GI inflammation, we have reviewed the clinical presentation, pathogenesis and current management of this disease. Increased understanding of the role of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex 2 (NOX2)-derived reactive oxygen species (ROS) in inflammatory bowel disease (IBD) will likely reveal novel targets for therapeutic intervention.
    Keywords:  Chronic granulomatous disease; Inflammatory bowel disease; Microbiome; NADPH oxidase; NOX2; Reactive oxygen species
    DOI:  https://doi.org/10.1007/978-1-4939-9424-3_34
  14. Methods Mol Biol. 2019 ;1982 447-458
    Protein-Protein Interaction Assay to Analyze NOX4/p22phox Heterodimerization.
    Sharon O'Neill, Ulla G Knaus.
      The stabilization and activation of NOX4 through its binding with p22phox are well documented; however little is known of the precise manner by which these two proteins interact. In recent years, the field of proteomics has undergone tremendous development with the introduction of many novel methods for the identification and characterization of protein-protein interactions (PPIs). To enhance our understanding of structural determinants leading to the association between NOX4 and p22phox, we developed a binary luciferase reporter assay (NanoBiT®) to quantitatively assess NOX4-p22phox heterodimerization. The complementation reporter quantitatively determines the accurate, reduced, or failed complex assembly, which can be confirmed and further interrogated by analyzing NOX4 catalytic activity (H2O2 release), protein expression, and dimer localization. This association-based PPI technique represents both a much-needed expansion of the NOX4 lead discovery tool box and a versatile method to probe the architecture of NOX and DUOX complexes in the future.
    Keywords:  Bioluminescence; Heterodimerization; NADPH oxidase; NOX4; NanoBiT®; Protein–protein interaction (PPI); p22phox
    DOI:  https://doi.org/10.1007/978-1-4939-9424-3_26
  15. Methods Mol Biol. 2019 ;1982 487-496
    NADPH Oxidases and Aging Models of Lung Fibrosis.
    Karen Bernard, Victor J Thannickal.
      There is a growing recognition that aging is a risk factor for fibrosis that affects a number of organ systems, including the lung. Despite this understanding, most studies of experimental fibrosis have been conducted in young mice that typically resolve injury-induced lung fibrosis over the course of several months. Our studies demonstrate that aged mouse models may recapitulate human disease by generating a more persistent fibrotic response to injury. This is, in part, due to an imbalance in the expression and activity of NADPH oxidase (NOX) enzymes, in particular the NOX4 isoform, and a related deficiency in antioxidant responses in pathogenic myofibroblasts. These pathogenic myofibroblasts acquire features of cellular senescence and become resistant to apoptosis. In this chapter, we present methods and procedures to apply the aging model of lung fibrosis in mice that will allow interrogation of myofibroblast functions and the expression and activity of NOX4 in cells. We provide recommendations for best laboratory practices to assess the severity and resolution of fibrosis in murine models of aging.
    Keywords:  Aging; Bleomycin injury; Fibrosis; Myofibroblasts; NADPH oxidase; NOX4; Oxidative stress
    DOI:  https://doi.org/10.1007/978-1-4939-9424-3_29
  16. Methods Mol Biol. 2019 ;1982 341-352
    Phosphorylation of gp91phox/NOX2 in Human Neutrophils.
    Houssam Raad, Riad Arabi Derkawi, Asma Tlili, Sahra A Belambri, Pham My-Chan Dang, Jamel El-Benna.
      The phagocyte NADPH oxidase NOX2 was the first NOX family member to be discovered. It is responsible for the production of reactive oxygen species that are required for bacterial killing and host defense. Activated NOX2 is an enzymatic complex composed of two membrane proteins, p22phox and gp91phox (renamed NOX2), which form the cytochrome b558, and four cytosolic proteins, p47phox, p67phox, p40phox, and the small GTPase Rac2. Except for Rac2, all proteins from the complex become phosphorylated during neutrophil activation, suggesting the importance of this process in NOX2 regulation. The phosphorylation of the cytosolic components, and in particular p47phox, has been extensively studied; however, the phosphorylation of the membrane proteins was less studied, in part due to the lack of good antibodies and accurate membrane solubilization techniques. In this chapter, we describe a method we have used to study NOX2 phosphorylation, which is based on the labeling of the intracellular ATP pool with 32P prior to applying a stimulus inducing protein phosphorylation. We also describe the solubilization of membrane-bound gp91phox/NOX2 and analysis by immunoprecipitation, polyacrylamide gel electrophoresis, electrophoretic transfer, phosphoamino acid analysis, and autoradiography. This protocol can also be used to study the possible phosphorylation of other NOX family members.
    Keywords:  NADPH oxidase; NOX2; Neutrophils; Protein phosphorylation; Respiratory burst; gp91phox
    DOI:  https://doi.org/10.1007/978-1-4939-9424-3_21
  17. Methods Mol Biol. 2019 ;1982 121-137
    Soluble Regulatory Proteins for Activation of NOX Family NADPH Oxidases.
    Hideki Sumimoto, Reiko Minakami, Kei Miyano.
      NOX family NADPH oxidases deliberately produce reactive oxygen species and thus contribute to a variety of biological functions. Of seven members in the human family, the three oxidases NOX2, NOX1, and NOX3 form a heterodimer with p22phox and are regulated by soluble regulatory proteins: p47phox, its related organizer NOXO1; p67phox, its related activator NOXA1; p40phox; and the small GTPase Rac. Activation of the phagocyte oxidase NOX2 requires p47phox, p67phox, and GTP-bound Rac. In addition to these regulators, p40phox plays a crucial role when NOX2 is activated during phagocytosis. On the other hand, NOX1 activation prefers NOXO1 and NOXA1, although Rac is also involved. NOX3 constitutively produces superoxide, which is enhanced by regulatory proteins such as p47phox, NOXO1, and p67phox. Here we describe mechanisms for NOX activation with special attention to the soluble regulatory proteins.
    Keywords:  NOX1; NOX2; NOX3; NOXA1; NOXO1; Rac; p22phox; p40phox; p47phox; p67phox
    DOI:  https://doi.org/10.1007/978-1-4939-9424-3_8
  18. Methods Mol Biol. 2019 ;1982 191-229
    Guidelines for the Detection of NADPH Oxidases by Immunoblot and RT-qPCR.
    Becky A Diebold, S Garrett Wilder, Xavier De Deken, Jennifer L Meitzler, James H Doroshow, James W McCoy, Yerun Zhu, J David Lambeth.
      The identification of NADPH oxidase (NOX) isoforms in tissues is essential for interpreting experiments and for next step decisions regarding cell lines, animal models, and targeted drug design. Two basic methods, immunoblotting and reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR), are important to monitor NOX protein and messenger RNA (mRNA) levels, respectively, for a range of investigations from understanding cell signaling events to judging NOX inhibitor efficacies. For many other genes that are expressed in high abundance, these methods may seem rather simple. However, detecting the low expression levels of endogenous NOX/DUOX is difficult and can be frustrating, so some guidelines would be helpful to those who are facing difficulties. One reason why detection is so difficult is the limited availability of vetted NOX/DUOX antibodies. Many of the commercial antibodies do not perform well in our hands, and dependable antibodies, often generated by academic laboratories, are in limited supply. Another problem is the growing trend in the NOX literature to omit end-user validation of antibodies by not providing appropriate positive and negative controls. With regard to NOX mRNA levels, knockdown of NOX/DUOX has been reported in cell lines with very low endogenous expression (C q values ≥30) or in cell lines devoid of the targeted NOX isoform (e.g., NOX4 expression in NCI-60 cancer cell panel cell line 786-0). These publications propagate misinformation and hinder progress in understanding NOX/DUOX function. This chapter provides overdue guidelines on how to validate a NOX antibody and provides general methodologies to prepare samples for optimal detection. It also includes validated methodology to perform RT-qPCR for the measurement of NOX mRNA levels, and we suggest that RT-qPCR should be performed prior to embarking on NOX protein detection.
    Keywords:  Immunoblot; NOX antibodies; NOX qPCR primers
    DOI:  https://doi.org/10.1007/978-1-4939-9424-3_12
  19. Methods Mol Biol. 2019 ;1982 3-16
    Intersecting Stories of the Phagocyte NADPH Oxidase and Chronic Granulomatous Disease.
    William M Nauseef, Robert A Clark.
      Neutrophils serve as the circulating cells that respond early and figure prominently in human host defense to infection and in inflammation in other settings. Optimal oxidant-dependent antimicrobial activity by neutrophils relies on the ability of stimulated phagocytes to utilize a multicomponent NADPH oxidase to generate oxidants. The frequent, severe, and often fatal infections experienced by individuals with chronic granulomatous disease (CGD), an inherited disorder in which one of the NADPH oxidase components is absent or dysfunctional, underscore the link between a functional phagocyte NADPH oxidase and robust host protection against microbial infection.The history of the discovery and characterization of the normal neutrophil NADPH oxidase and the saga of recognizing CGD and its underlying causes together illustrate how the observations of astute clinicians and imaginative basic scientists synergize to forge new understanding of both basic cell biology and pathogenesis of human disease.In this chapter, we review the events in the stepwise evolution of our understanding of the phagocyte NADPH oxidase, both in the context of normal human neutrophil function and in the setting of CGD. The phagocyte oxidase complex employs a heterodimeric transmembrane protein composed of gp91phox and p22phox to relay electrons from NADPH to molecular oxygen, while other cofactors contribute to localization and regulation of the activity of the assembled oxidase. The b-type cytochrome gp91phox, also known as NOX2, serves as the catalytic component of this multicomponent enzyme complex. Although many of the features of the composition and regulation of the phagocyte oxidase may apply as well to NOX2 expressed in non-phagocytes and to other members of the NOX protein family, exceptions exist and pose special challenges to investigators exploring the biology of NADPH oxidases.
    Keywords:  Chronic granulomatous disease (CGD); Cytochrome b558; NOX2; Phagocyte NADPH oxidase; gp91phox; p22phox; p40phox; p47phox; p67phox
    DOI:  https://doi.org/10.1007/978-1-4939-9424-3_1
  20. Methods Mol Biol. 2019 ;1982 153-171
    The X-CGD PLB-985 Cell Model for NOX2 Structure-Function Analysis.
    Sylvain Beaumel, Marie José Stasia.
      Structure-function analysis of specific regions of NOX2 can be carried out after stable expression of site-directed mutagenesis-modified NOX2 in the X0-CGD PLB-985 cell model. Indeed, the generation of this human cellular model by Prof. MC Dinauer's team gave researchers the opportunity to gain a deeper understanding of functional regions of NOX2. With this model cell line, the functional impact of X+-CGD or of new mutations in NOX2 can be highlighted, as the biological material is not limited. PLB-985 cells transfected with various NOX2 mutations can be easily cultured and differentiated into neutrophils or monocytes/macrophages. Several measurements in intact mutated NOX2 PLB-985 cells can be carried out such as NOX2 expression, cytochrome b 558 spectrum, enzymatic activity, and assembly of the NADPH oxidase complex. Purified membranes or purified cytochrome b 558 from mutated NOX2 PLB-985 cells can be used for the study of the impact of specific mutations on NADPH oxidase or diaphorase activity, FAD incorporation, and NADPH or NADH binding in a cell-free assay system. Here, we describe a method to generate mutated NOX2 PLB-985 cells in order to analyze NOX2 structure-function relationships.
    Keywords:  Cellular model; Chronic granulomatous disease; NADPH oxidase activity; NOX2 deficiency; PLB-985 cell line; Structure-function analysis
    DOI:  https://doi.org/10.1007/978-1-4939-9424-3_10
  21. Methods Mol Biol. 2019 ;1982 61-74
    Purification and Characterization of DUOX Peroxidase Homology Domains (PHDs).
    Jennifer L Meitzler.
      The dual oxidase (DUOX) enzymes (DUOX1 and DUOX2) are unique hydrogen peroxide (H2O2)-producing members of the NADPH oxidase (NOX) family, structurally distinguished from their related NOX isoforms by the presence of an additional N-terminal extracellular domain. This region has significant sequence and predicted structural homology to mammalian peroxidases, including myeloperoxidase (MPO) and lactoperoxidase (LPO), therefore justifying the nomenclature of the peroxidase homology domain (PHD). Obtaining detailed structural information and defining a function for this appended region are both critical for elucidation of the uncharacterized mechanism of H2O2 production by DUOX proteins. Purification strategies focused on isolated sections of each DUOX enzyme are a logical means to further characterization, particularly as isolation of the complete membrane-bound enzyme in significant quantities remains unachievable. In this chapter, a reproducible method for production of the homology domain applicable to both human DUOX isoforms is described. The approach utilizes a baculovirus expression vector in insect cell culture to produce secreted recombinant PHD; an appended C-terminal His6 affinity tag was found to be crucial for structural stability. Finally, initial characterization of the activity of the purified PHDs is also described.
    Keywords:  Baculovirus; Dual oxidase; Hydrogen peroxide; Mammalian peroxidase
    DOI:  https://doi.org/10.1007/978-1-4939-9424-3_4
  22. Methods Mol Biol. 2019 ;1982 103-111
    NOX5 Cell-Free Assay for the High-Throughput Screening of Small Molecules.
    Fiona Augsburger, Delphine Rasti, Yves Cambet, Vincent Jaquet.
      NADPH oxidases (NOX) are a family of transmembrane enzymes, which catalyze the formation of O2 ˙- and H2O2. Membrane fractions of leukocytes are highly enriched in the phagocyte NOX isoform (NOX2). This feat has allowed the development of a complex NOX2 cell-free assay, which has been a key tool for the understanding of the mode of action of NOX2, its biochemistry, pharmacology, and identification of NOX2-specific inhibitors. In addition to NOX2, there are six other NOX isoforms in humans, but cell-free assays of non-phagocytic oxidases are infrequently used, and their specificity has recently been debated. Here we describe a NOX5 cell-free assay. We present a method to purify the membranous component of cells stably transduced with NOX5 and to measure O2 ˙- in a high-throughput format (96-w or 384-w plates). The experimental description allows high-throughput screening of small molecules with limited cost.
    Keywords:  Cell-free assay; High-throughput screening; Membrane purification; NOX5
    DOI:  https://doi.org/10.1007/978-1-4939-9424-3_6
  23. Methods Mol Biol. 2019 ;1982 429-446
    High-Throughput Screening of NOX Inhibitors.
    Jacek Zielonka, Monika Zielonka, Gang Cheng, Micael Hardy, Balaraman Kalyanaraman.
      Development of new, selective inhibitors of nicotinamide adenine dinucleotide phosphate oxidase (NOX) isoforms is important both for basic studies on the role of these enzymes in cellular redox signaling, cell physiology, and proliferation and for development of new drugs for diseases carrying a component of increased NOX activity, such as several types of cancer and cardiovascular and neurodegenerative diseases. High-throughput screening (HTS) of large libraries of compounds remains the major approach for development of new NOX inhibitors. Here, we describe the protocol for the HTS campaign for NOX inhibitors using rigorous assays for superoxide radical anion and hydrogen peroxide, based on oxidation of hydropropidine, coumarin boronic acid, and Amplex Red. We propose using these three probes to screen for and identify new inhibitors, by selecting positive hits that show inhibitory effects in all three assays. Protocols for the synthesis of hydropropidine and for confirmatory assays, including oxygen consumption measurements, electron paramagnetic resonance spin trapping of superoxide, and simultaneous monitoring of superoxide and hydrogen peroxide, are also provided.
    Keywords:  Amplex Red; Coumarin boronic acid; EPR spin trapping; Fluorescence; High-throughput screening; Hydrogen peroxide; Hydropropidine; Seahorse extracellular flux analyzer; Superoxide radical anion
    DOI:  https://doi.org/10.1007/978-1-4939-9424-3_25
  24. Methods Mol Biol. 2019 ;1982 139-151
    Insights into the NOX NADPH Oxidases Using Heterologous Whole Cell Assays.
    Mary C Dinauer.
      Assays based on ectopic expression of NOX NADPH oxidase subunits in heterologous mammalian cells are an important approach for investigating features of this family of enzymes. These model systems have been used to analyze the biosynthesis and functional domains of NOX enzyme components as well as their regulation and cellular activities. This chapter provides an overview of the basic principles and applications of heterologous whole cell assays in studying NOX NADPH oxidases.
    Keywords:  COS-7; Expression; Flavocytochrome b; NOX; phox
    DOI:  https://doi.org/10.1007/978-1-4939-9424-3_9
  25. Methods Mol Biol. 2019 ;1982 417-428
    Rational Design and Delivery of NOX-Inhibitory Peptides.
    Eugenia Cifuentes-Pagano, Patrick J Pagano.
      A growing appreciation of NADPH oxidases (NOXs) as mediators of fundamental physiological processes and as important players in myriad diseases has led many laboratories on a search for specific inhibitors to help dissect the role in a given pathway or pathological condition. To date, there are only a few available inhibitors with a demonstrated specificity for a given isozyme. Among those, peptidic inhibitors have the advantage of being designed to target very specific protein-protein interactions that are essential for NOX activity. Herein, we provide the techniques to deliver these inhibitors both in cell culture as well as in vivo.
    Keywords:  Isoform-specific inhibitors; NADPH oxidase; Nox2ds-tat; NoxA1ds; Peptidic inhibitors
    DOI:  https://doi.org/10.1007/978-1-4939-9424-3_24
  26. Methods Mol Biol. 2019 ;1982 113-120
    Spectroscopy of NOX Protein Family Members.
    Yoko Nakano, William M Nauseef.
      All members of the NOX protein family contain a unique b-type cytochrome that mediates the electron transport that characterizes the activity of the multicomponent oxidase complexes. Referred to as cytochrome b558, because of its signature spectral absorbance at 558 nm in reduced-minus-oxidized difference spectroscopy, or cytochrome b(-245), because of its very low midpoint potential of -245 mV at pH 7.0, the protein possesses two stacked inequivalent hemes ligated by pairs of histidine residues in membrane helices h3 and h5. In a flavin-dependent fashion, cytochrome b558 shuttles electrons from cytoplasmic NADPH across membranes to molecular oxygen and thereby generates superoxide anion. By performing reduced-minus-oxidized difference spectroscopy and using the millimolar extinction coefficient, E 559-540 nm = 21.6 cm-1 mM-1, one can calculate the amount of cytochrome b558 in intact cells or partially purified membrane preparations. Measurements in samples where cytochrome b558 is relatively high and the presence of unrelated heme-containing proteins low, as in neutrophils, are straightforward. However, low levels of cytochrome b558 expression combined with an abundance of mitochondria and other sources of heme proteins make spectral detection of cytochrome b558 in non-phagocytic cells extremely challenging.
    Keywords:  Cytochrome b(-245); Cytochrome b558; Dithionite-reduced-minus-oxidized spectroscopy; NOX proteins; gp91phox
    DOI:  https://doi.org/10.1007/978-1-4939-9424-3_7
  27. Methods Mol Biol. 2019 ;1982 173-190
    Functional Characterization of DUOX Enzymes in Reconstituted Cell Models.
    Agnieszka Korzeniowska, Ágnes P Donkó, Stanislas Morand, Thomas L Leto.
      Biosynthesis of active human dual oxidases (DUOX1 and DUOX2) requires maturation factors, a.k.a. DUOX activator proteins (DUOXA1 and DUOXA2), that form covalent complexes with DUOX; both chains together represent the mature catalytic unit that functions as a dedicated hydrogen peroxide-generating enzyme. Genetic defects in DUOX2 or DUOXA2 can result in congenital hypothyroidism, whereas partial defects in DUOX2 activity also have been associated with very early-onset inflammatory bowel disease. Our understanding of the links between DUOX dysfunction and these diseases remains incomplete. An important challenge in developing a better understanding of the pathogenic roles of DUOX defects requires robust and reliable DUOX reconstitution cell models to examine the functional consequences of candidate DUOX missense mutations and polymorphisms. Here, we describe methods for efficient heterologous DUOX/DUOXA co-expression and functional characterization, including detailed assessments of posttranslational processing and subcellular translocation of DUOX that accompanies the maturation of these enzymes into catalytically active NADPH oxidases.
    Keywords:  Cell surface targeting; DUOX; DUOX activator; DUOXA; Dual oxidases; Hydrogen peroxide; NADPH oxidases; NOX
    DOI:  https://doi.org/10.1007/978-1-4939-9424-3_11
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