186 results on '"Leto, Thomas L."'
Search Results
152. The Interaction of Cytochrome b5 with Lipid Vesicles
- Author
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Holloway, Peter W., Markello, Tom C., and Leto, Thomas L.
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- 1982
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153. Lipids and proteins in the Rathke's gland secretions of the North American mud turtle ( Kinosternon subrubrum)
- Author
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Seifert, William E., Jr, Gotte, Steve W., Leto, Thomas L., and Weldon, Paul J.
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- 1994
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154. Plant and human neutrophil oxidative burst complexes contain immunologically related proteins
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Dwyer, Shannon C., Legendre, Laurent, Low, Philip S., and Leto, Thomas L.
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- 1996
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155. Retroviral Expression of Recombinant p47phox Protein by Epstein-Barr Virus-Transformed B Lymphocytes From a Patient With Autosomal Chronic Granulomatous Disease
- Author
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Cobbs, Charles S., Malech, Harry L., Leto, Thomas L., Freeman, Scott M., Blaese, R. Michael, Gallin, John I., and Lomax, Karen J.
- Published
- 1992
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156. Characterization of the p67phox Gene: Genomic Organization and Restriction Fragment Length Polymorphism Analysis for Prenatal Diagnosis in Chronic Granulomatous Disease
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Kenney, Richard T., Malech, Harry L., Epstein, Neal D., Roberts, Robert L., and Leto, Thomas L.
- Published
- 1993
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157. The N-formylpeptide receptor (FPR) and a second <f>Gi</f>-coupled receptor mediate fMet–Leu–Phe-stimulated activation of NADPH oxidase in murine neutrophils
- Author
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Lavigne, Mark C., Murphy, Philip M., Leto, Thomas L., and Gao, Ji-Liang
- Subjects
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PEPTIDES , *CELLULAR signal transduction - Abstract
N-Formylypeptides such as fMet–Leu–Phe (fMLF) potently induce superoxide production through NADPH oxidase activation. The receptors that mediate this response have not been defined. Here, we provide definitive proof using a mouse model that formyl peptide receptor (FPR) is a receptor, but not the only receptor, that mediates fMLF-induced oxidase activation. In wild-type (
FPR+/+ ) mouse neutrophils, superoxide production is dependent on the concentration of fMLF with anEC50 of∼ 5μM and a peak at∼ 50μM . In contrast, FPR-deficient (FPR−/− ) mouse neutrophils produced markedly less superoxide with anEC50 of∼ 50μM and a peak at∼ 200μM . Yet,FPR+/+ andFPR−/− neutrophils showed similar oxidase activation kinetics andGi protein-dependent pharmacological sensitivities. These results suggested that a second receptor, likely FPR2, mediates superoxide production at high concentrations of fMLF. This less sensitive second pathway may permit continued oxidant generation in response to formyl peptides when FPR is desensitized in high concentrations of the chemotactic gradient. [Copyright &y& Elsevier]- Published
- 2002
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158. Duox1 Regulates Primary B Cell Function under the Influence of IL-4 through BCR-Mediated Generation of Hydrogen Peroxide.
- Author
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Ryuichi Sugamata, Agnes Donko, Boudreau, Howard E., Jaeyul Kwon, Leto, Thomas L., Yousuke Murakami, and Chen-Feng Qi
- Abstract
Engagement of the BCR with Ags triggers signaling pathways for commitment of B lymphocyte responses that can be regulated, in part, by reactive oxygen species. To investigate the functional relevance of reactive oxygen species produced in primary B cells, we focused on the role of the hydrogen peroxide generator Duox1 in stimulated splenic B cells under the influence of the TH2 cytokine IL-4. We found that H2O2 production in wild type (WT) and Nox2-deficient CD19+ B cells was boosted concomitantly with enhanced expression of Duox1 following costimulation with BCR agonists together with IL-4, whereas stimulated Duox1-/- cells showed attenuated H2O2 release. We examined whether Duox1-derived H2O2 contributes to proliferative activity and Ig isotype production in CD19+ cells upon BCR stimulation. Duox1-/- CD19+ B cells showed normal responses of Ig production but a higher rate of proliferation than WTor Nox2-deficient cells. Furthermore, we demonstrated that the H2O2 scavenger catalase mimics the effect of Duox1 deficiency by enhancing proliferation of WT CD19+ B cells in vitro. Results from immunized mice reflected the in vitro observations: T cell-independent Ag induced increased B cell expansion in germinal centers from Duox1-/- mice relative to WTand Nox2-/- mice, whereas immunization with T cell-dependent or -independent Ag elicited normal Ig isotype secretion in the Duox1 mutant mice. These observations, obtained both by in vitro and in vivo approaches, strongly suggest that Duox1-derived hydrogen peroxide negatively regulates proliferative activity but not Ig isotype production in primary splenic CD19+ B cells. [ABSTRACT FROM AUTHOR]
- Published
- 2019
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159. Release of Cystic Fibrosis Airway Inflammatory Markers from Pseudomonas aeruginosa-Stimulated Human Neutrophils Involves NADPH Oxidase-Dependent Extracellular DNA Trap Formation.
- Author
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Dae-goon Yoo, Winn, Matthew, Lan Pang, Moskowitz, Samuel M., Malech, Harry L., Leto, Thomas L., and Rada, Balázs
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CYSTIC fibrosis , *NEUTROPHILS , *PSEUDOMONAS aeruginosa , *NADPH oxidase , *DNA , *MYELOPEROXIDASE , *AIRWAY (Anatomy) - Abstract
Cystic fibrosis (CF) airways are characterized by bacterial infections, excess mucus production, and robust neutrophil recruitment. The main CF airway pathogen is Pseudomonas aeruginosa. Neutrophils are not capable of clearing the infection. Neutrophil primary granule components, myeloperoxidase (MPO) and human neutrophil elastase (HNE), are inflammatory markers in CF airways, and their increased levels are associated with poor lung function. Identifying the mechanism of MPO and HNE release from neutrophils is of high clinical relevance for CF. In this article, we show that human neutrophils release large amounts of neutrophil extracellular traps (NETs) in the presence of P. aeruginosa. Bacteria are entangled in NETs and colocalize with extracellular DNA. MPO, HNE, and citrullinated histone H4 are all associated with DNA in Pseudomonas-triggered NETs. Both laboratory standard strains and CF isolates of P. aeruginosa induce DNA, MPO, and HNE release from human neutrophils. The increase in peroxidase activity of neutrophil supernatants after Pseudomonas exposure indicates that enzymatically active MPO is released. P. aeruginosa induces a robust respiratory burst in neutrophils that is required for extracellular DNA release. Inhibition of the cytoskeleton prevents Pseudomonas-initiated superoxide production and DNA release. NADPH oxidase inhibition suppresses Pseudomonas-induced release of active MPO and HNE. Blocking MEK/ERK signaling results in only minimal inhibition of DNA release induced by Pseudomonas. Our data describe in vitro details of DNA, MPO, and HNE release from neutrophils activated by P. aeruginosa. We propose that Pseudomonas-induced NET formation is an important mechanism contributing to inflammatory conditions characteristic of CF airways. [ABSTRACT FROM AUTHOR]
- Published
- 2014
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160. Cooperation of p40phox with p47phox for Nox2-based NADPH Oxidase Activation during Fcγ Receptor (FcγR)-mediated Phagocytosis: MECHANISM FOR ACQUISITION OF p40phox PHOSPHATIDYLINOSITOL 3-PHOSPHATE (PI(3)P) BINDING
- Author
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Ueyama, Takehiko, Nakakita, Junya, Nakamura, Takashi, Kobayashi, Takeshi, Kobayashi, Toshihiro, Jeonghyun Son, Sakuma, Megumi, Sakaguchi, Hirofumi, Leto, Thomas L., and Saito, Naoaki
- Subjects
- *
PHOSPHOINOSITIDES , *PHAGOCYTES , *OXIDASES , *PHAGOCYTOSIS , *CYTOPLASM - Abstract
During activation of the phagocyte (Nox2-based) NADPH oxidase, the cytoplasmic Phox complex (p47phox-p67phox-p40phox) translocates and associates with the membrane-spanning flavocytochrome b558. It is unclear where (in cytoplasm or on membranes), when (before or after assembly), and how p40phox acquires its PI(3)P-binding capabilities. We demonstrated that in addition to conformational changes induced by H2O2 in the cytoplasm, p40phox acquires PI(3)P-binding through direct or indirect membrane targeting. We also found that p40phox is essential when p47phox is partially phosphorylated during FcγR-mediated oxidase activation; however, p40phox is less critical when p47phox is adequately phosphorylated, using phosphorylation-mimicking mutants in HEK293Nox2/FcγRIIa and RAW264.7p40/p47KD cells. Moreover, PI binding to p47phox is less important when the autoinhibitory PX-PB1 domain interaction in p40phox is disrupted or when p40phox is targeted to membranes. Furthermore, we suggest that high affinity PI(3)P binding of the p40phox PX domain is critical during its accumulation on phagosomes, even when masked by the PB1 domain in the resting state. Thus, in addition to mechanisms for directly acquiring PI(3)P binding in the cytoplasm by H2O2, p40phox can acquire PI(3)P binding on targeted membranes in a p47phox-dependent manner and functions both as a "carrier" of the cytoplasmic Phox complex to phagosomes and an "adaptor" of oxidase assembly on phagosomes in cooperation with p47phox, using positive feedback mechanisms. [ABSTRACT FROM AUTHOR]
- Published
- 2011
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161. Double-Stranded RNA Induces Shedding of the 34-kDa Soluble TNFR1 from Human Airway Epithelial Cells via TLR3-TRIF-RIP1-Dependent Signaling: Roles for Dual Oxidase 2- and Caspase-Dependent Pathways.
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Man Yu, Lam, Jonathan, Rada, Balázs, Leto, Thomas L., and Levine, Stewart J.
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TUMOR necrosis factors , *IMMUNE response , *EPITHELIAL cells , *REACTIVE oxygen species , *APOPTOSIS - Abstract
TNF, an important mediator of inflammatory and innate immune responses, can be regulated by binding to soluble TNF receptors. The 55-kDa type 1 TNFR (TNFR1), the key receptor for TNF signaling, is released to the extracellular space by two mechanisms, the inducible cleavage and shedding of 34-kDa soluble TNFR1 (sTNFR1) ectodomains and the constitutive release of full-length 55-kDa TNFR1 within exosome-like vesicles. The aim of this study was to identify and characterize TLR signaling pathways that mediate TNFR1 release to the extracellular space. To our knowledge, we demonstrate for the first time that polyinosinic-polycytidylic acid [poly (I:C)], a synthetic dsRNA analogue that signals via TLR3, induces sTNFR1 shedding from human airway epithelial (Nd- H292) cells, whereas ligands for other microbial pattern recognition receptors, including TLR4, TLR7, and nucleotide-binding oligomerization domain containing 2, do not. Furthermore, poly (I:C) selectively induces the cleavage of 34-kDa sTNFR1 ecto- domains but does not enhance the release of full-length 55-kDa TNFR1 within exosome-like vesicles. RNA interference experi- ments demonstrated that poly (I:C)-induced sTNFR1 shedding is mediated via activation of TLR3-TRIF-RIP1 signaling, with subsequent activation of two downstream pathways. One pathway involves the dual oxidase 2-mediated generation of reactive oxygen species, and the other pathway is via the caspase-mediated activation of apoptosis. Thus, the ability of dsRNA to induce the cleavage and shedding of the 34-kDa sTNFR1 from human bronchial epithelial cells represents a novel mechanism by which innate immune responses to viral infections are modulated. [ABSTRACT FROM AUTHOR]
- Published
- 2011
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162. Cytosolic Phospholipase A2α Is Targeted to the p47phox-PX Domain of the Assembled NADPH Oxidase via a Novel Binding Site in Its C2 Domain.
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Shmelzer, Zeev, Karter, Maria, Eisenstein, Miriam, Leto, Thomas L., Hadad, Nurit, Ben-Menahem, David, Gitler, Daniel, Banani, Shirly, Wolach, Baruch, Rotem, Meir, and Levy, Rachel
- Subjects
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PHOSPHOLIPASES , *CELL membranes , *BINDING sites , *ENZYMES , *NEUTROPHILS , *BIOCHEMISTRY - Abstract
We have previously demonstrated a physical interaction between cytosolic phospholipase A2α (cPLA2) and the assembled NADPH oxidase on plasma membranes following neutrophil stimulation. The aim of the present study was to define the exact binding sites between these two enzymes. Here we show, based on blot overlay experiments, Förster resonance energy transfer analysis and studies in neutrophils from patients with chronic granulomatous disease deficient in p67phox or p47phox, that cPLA2 specifically binds to p47phox and that p47phox is sufficient to anchor cPLA2 to the assembled oxidase on the plasma membranes upon stimulation. Blot overlay and affinity binding experiments using subfragments of cPLA2 and p47phox demonstrated that the cPLA2-C2 domain and the p47phox-PX domain interact to form a complex that is resistant to high salt. Computational docking was used to identify hydrophobic peptides within these two domains that inhibited the association between the two enzymes and NADPH oxidase activity in electro-permeabilized neutrophils. These results were used in new docking computations that produced an interaction model. Based on this model, cPLA2-C2 domain mutations were designed to explore its interaction p47phox in neutrophil lysates. The triple mutant F35A/M38A/L39A of the cPLA2-C2 domain caused a slight inhibition of the affinity binding to p47phox, whereas the single mutant I67A was highly effective. The double mutant M59A/H115A of the p47phox-PX domain caused a significant inhibition of the affinity binding to cPLA2. Thus, Ile67 of the cPLA2-C2 domain is identified as a critical, centrally positioned residue in a hydrophobic interaction in the p47phox-PX domain. [ABSTRACT FROM AUTHOR]
- Published
- 2008
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163. Delineation of the phagocyte NADPH oxidase through studies of chronic granulomatous diseases of childhood
- Author
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Gallin, John I., Leto, Thomas L., Rotrosen, Daniel, Kwong, Cheung H., and Malech, Harry L.
- Published
- 1992
- Full Text
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164. Functional Characterization of DUOX Enzymes in Reconstituted Cell Models.
- Author
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Korzeniowska A, Donkó ÁP, Morand S, and Leto TL
- Subjects
- Dual Oxidases chemistry, Dual Oxidases genetics, Enzyme Activation, Flow Cytometry, Fluorescent Antibody Technique, Gene Expression, HEK293 Cells, Humans, Hydrogen Peroxide metabolism, Protein Multimerization, Protein Transport, Dual Oxidases metabolism
- Abstract
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.
- Published
- 2019
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165. Model Systems to Investigate NOX-Dependent Cell Migration and Invasiveness.
- Author
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Boudreau HE and Leto TL
- Subjects
- Animals, Biomarkers, Cell Culture Techniques, Cell Line, Tumor, Humans, Oxidation-Reduction, Reactive Oxygen Species metabolism, Wound Healing, Cell Movement, NADPH Oxidases metabolism
- Abstract
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.
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- 2019
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166. Pyocyanin effects on respiratory epithelium: relevance in Pseudomonas aeruginosa airway infections.
- Author
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Rada B and Leto TL
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- Cystic Fibrosis metabolism, Cystic Fibrosis microbiology, Gene Expression Regulation, Host-Pathogen Interactions, Humans, Mucins biosynthesis, Oxidation-Reduction, Pseudomonas Infections genetics, Pseudomonas aeruginosa pathogenicity, Virulence Factors, Pseudomonas Infections metabolism, Pseudomonas aeruginosa metabolism, Pyocyanine metabolism, Respiratory Mucosa metabolism, Respiratory Mucosa microbiology
- Abstract
Pseudomonas aeruginosa (PA) uses several virulence factors to establish chronic respiratory infections in bronchiectasis, chronic obstructive pulmonary disease, and cystic fibrosis (CF) patients. One of its toxins, pyocyanin (PYO), is a redox-active pigment that is required for full virulence in animal models and has been detected in patients' airway secretions. PYO promotes virulence by interfering with several cellular functions in host cells including electron transport, cellular respiration, energy metabolism, gene expression, and innate immune mechanisms. This review summarizes recent advances in PYO biology with special attention to current views on its role in human airway infections and on its interactions with the first line of our airway defense, the respiratory epithelium., (Published by Elsevier Ltd.)
- Published
- 2013
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167. Nox4 involvement in TGF-beta and SMAD3-driven induction of the epithelial-to-mesenchymal transition and migration of breast epithelial cells.
- Author
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Boudreau HE, Casterline BW, Rada B, Korzeniowska A, and Leto TL
- Subjects
- Cell Line, Tumor, Cell Movement, Cell Proliferation, Epithelial Cells cytology, Epithelial Cells drug effects, Epithelial-Mesenchymal Transition drug effects, Female, Fibronectins genetics, Fibronectins metabolism, Gene Expression Regulation, Humans, Isoquinolines pharmacology, Mammary Glands, Human cytology, NADPH Oxidase 4, NADPH Oxidases genetics, Pyridines pharmacology, Pyrroles pharmacology, RNA, Small Interfering genetics, Reactive Oxygen Species metabolism, Receptors, Transforming Growth Factor beta genetics, Receptors, Transforming Growth Factor beta metabolism, Signal Transduction, Smad3 Protein antagonists & inhibitors, Smad3 Protein genetics, Transforming Growth Factor beta pharmacology, Epithelial Cells metabolism, Epithelial-Mesenchymal Transition genetics, Mammary Glands, Human metabolism, NADPH Oxidases metabolism, Smad3 Protein metabolism, Transforming Growth Factor beta metabolism
- Abstract
The epithelial-to-mesenchymal transition (EMT) is the development of increased cell plasticity that occurs normally during wound healing and embryonic development and can be coopted for cancer invasion and metastasis. TGF-beta induces EMT but the mechanism is unclear. Our studies suggest that Nox4, a member of the NADPH oxidase (Nox) family, is a source of reactive oxygen species (ROS) affecting cell migration and fibronectin expression, an EMT marker, in normal and metastatic breast epithelial cells. We found that TGF-beta induces Nox4 expression (mRNA and protein) and ROS generation in normal (MCF10A) and metastatic (MDA-MB-231) human breast epithelial cells. Conversely, cells expressing a dominant-negative form of Nox4 or Nox4-targeted shRNA showed significantly lower ROS production on TGF-beta treatment. Expression of a constitutively active TGF-beta receptor type I significantly increased Nox4 promoter activity, mRNA and protein expression, and ROS generation. Nox4 transcriptional regulation by TGF-beta was SMAD3 dependent based on the effect of constitutively active SMAD3 increasing Nox4 promoter activity, whereas dominant-negative SMAD3 or SIS3, a SMAD3-specific inhibitor, had the opposite effect. Furthermore, Nox4 knockdown, dominant-negative Nox4 or SMAD3, or SIS3 blunted TGF-beta induced wound healing and cell migration, whereas cell proliferation was not affected. Our experiments further indicate that Nox4 plays a role in TGF-beta regulation of fibronectin mRNA expression, based on the effects of dominant-negative Nox4 in reducing fibronectin mRNA in TGF-beta-treated MDA-MB-231and MCF10A cells. Collectively, these data indicate that Nox4 contributes to NADPH oxidase-dependent ROS production that may be critical for the progression of the EMT in breast epithelial cells, and thereby has therapeutic implications., (Published by Elsevier Inc.)
- Published
- 2012
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168. Plasmodium falciparum merozoite surface protein 1 blocks the proinflammatory protein S100P.
- Author
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Waisberg M, Cerqueira GC, Yager SB, Francischetti IM, Lu J, Gera N, Srinivasan P, Miura K, Rada B, Lukszo J, Barbian KD, Leto TL, Porcella SF, Narum DL, El-Sayed N, Miller LH, and Pierce SK
- Subjects
- Amino Acid Sequence, Animals, Calcium-Binding Proteins chemistry, Chromatography, Gel, Electrophoresis, Polyacrylamide Gel, Enzyme-Linked Immunosorbent Assay, Humans, Microscopy, Confocal, Molecular Sequence Data, Neoplasm Proteins chemistry, Sequence Homology, Amino Acid, Surface Plasmon Resonance, Calcium-Binding Proteins antagonists & inhibitors, Merozoite Surface Protein 1 physiology, Neoplasm Proteins antagonists & inhibitors, Plasmodium falciparum metabolism
- Abstract
The malaria parasite, Plasmodium falciparum, and the human immune system have coevolved to ensure that the parasite is not eliminated and reinfection is not resisted. This relationship is likely mediated through a myriad of host-parasite interactions, although surprisingly few such interactions have been identified. Here we show that the 33-kDa fragment of P. falciparum merozoite surface protein 1 (MSP1(33)), an abundant protein that is shed during red blood cell invasion, binds to the proinflammatory protein, S100P. MSP1(33) blocks S100P-induced NFκB activation in monocytes and chemotaxis in neutrophils. Remarkably, S100P binds to both dimorphic alleles of MSP1, estimated to have diverged >27 Mya, suggesting an ancient, conserved relationship between these parasite and host proteins that may serve to attenuate potentially damaging inflammatory responses.
- Published
- 2012
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169. Peroxiredoxin 6 translocates to the plasma membrane during neutrophil activation and is required for optimal NADPH oxidase activity.
- Author
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Ambruso DR, Ellison MA, Thurman GW, and Leto TL
- Subjects
- Humans, K562 Cells, NADPH Oxidases chemistry, NADPH Oxidases genetics, Neutrophils cytology, Peroxiredoxin VI genetics, Protein Isoforms genetics, Protein Isoforms metabolism, Protein Subunits chemistry, Protein Subunits genetics, Protein Subunits metabolism, RNA, Small Interfering genetics, RNA, Small Interfering metabolism, Transgenes, Cell Membrane metabolism, NADPH Oxidases metabolism, Neutrophil Activation, Neutrophils metabolism, Peroxiredoxin VI metabolism
- Abstract
Neutrophils provide the first line of defense against microbial invasion in part through production of reactive oxygen species (ROS) which is mediated through activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase generating superoxide anion (O2-). The phagocyte oxidase (phox) has multiple protein components that assemble on the plasma membrane in stimulated neutrophils. We recently described a protein in neutrophils, peroxiredoxin 6 (Prdx6), which has both peroxidase and phospholipase A2 (PLA2) activities and enhances oxidase activity in an SDS-activated, cell-free system. The function of Prdx6 in phox activity is further investigated. In reconstituted phox-competent K562 cells, siRNA-mediated suppression of Prdx6 resulted in decreased NADPH oxidase activity in response to formyl-methionyl-leucyl-phenylalanine (fMLP) or phorbol myristate acetate (PMA). In neutrophils stimulated with PMA, Prdx6 translocated to plasma membrane as demonstrated by Western blot and confocal microscopy. Translocation of Prdx6 in phox competent K562 cells required both p67phox and p47phox. In addition, plasma membrane from PMA-stimulated, oxidase competent K562 cells with siRNA-mediated Prdx6 suppression contained less p47phox and p67phox compared to cells in which Prdx6 was not decreased. Cell-free oxidase assays showed that recombinant Prdx6 did not alter the Km for NADPH, but increased the Vmax for O2- production in a saturable, Prdx6 concentration-dependent manner. Recombinant proteins with mutations in Prdx (C47S) and phospholipase (S32A) activity both enhanced cell-free phox activity to the same extent as wild type protein. Prdx6 supports retention of the active oxidase complex in stimulated plasma membrane, and results with mutant proteins imply that Prdx6 serves an additional biochemical or structural role in supporting optimal NADPH oxidase activity., (Copyright © 2011 Elsevier B.V. All rights reserved.)
- Published
- 2012
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170. Cooperation of p40(phox) with p47(phox) for Nox2-based NADPH oxidase activation during Fcγ receptor (FcγR)-mediated phagocytosis: mechanism for acquisition of p40(phox) phosphatidylinositol 3-phosphate (PI(3)P) binding.
- Author
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Ueyama T, Nakakita J, Nakamura T, Kobayashi T, Kobayashi T, Son J, Sakuma M, Sakaguchi H, Leto TL, and Saito N
- Subjects
- Animals, Cell Membrane drug effects, Cell Membrane metabolism, Endosomes drug effects, Endosomes metabolism, Enzyme Activation drug effects, Glass chemistry, HEK293 Cells, Humans, Hydrogen Peroxide pharmacology, Immunoglobulin G chemistry, Immunoglobulin G pharmacology, Mice, Mutation, NADPH Oxidase 2, NADPH Oxidases chemistry, Phagosomes drug effects, Phagosomes metabolism, Phosphoproteins chemistry, Phosphoproteins genetics, Phosphorylation drug effects, Protein Binding, Protein Structure, Tertiary, Protein Transport drug effects, Reactive Oxygen Species metabolism, Respiratory Burst drug effects, Membrane Glycoproteins metabolism, NADPH Oxidases metabolism, Phagocytosis drug effects, Phosphatidylinositol Phosphates metabolism, Phosphoproteins metabolism, Receptors, IgG metabolism
- Abstract
During activation of the phagocyte (Nox2-based) NADPH oxidase, the cytoplasmic Phox complex (p47(phox)-p67(phox)-p40(phox)) translocates and associates with the membrane-spanning flavocytochrome b(558). It is unclear where (in cytoplasm or on membranes), when (before or after assembly), and how p40(phox) acquires its PI(3)P-binding capabilities. We demonstrated that in addition to conformational changes induced by H(2)O(2) in the cytoplasm, p40(phox) acquires PI(3)P-binding through direct or indirect membrane targeting. We also found that p40(phox) is essential when p47(phox) is partially phosphorylated during FcγR-mediated oxidase activation; however, p40(phox) is less critical when p47(phox) is adequately phosphorylated, using phosphorylation-mimicking mutants in HEK293(Nox2/FcγRIIa) and RAW264.7(p40/p47KD) cells. Moreover, PI binding to p47(phox) is less important when the autoinhibitory PX-PB1 domain interaction in p40(phox) is disrupted or when p40(phox) is targeted to membranes. Furthermore, we suggest that high affinity PI(3)P binding of the p40(phox) PX domain is critical during its accumulation on phagosomes, even when masked by the PB1 domain in the resting state. Thus, in addition to mechanisms for directly acquiring PI(3)P binding in the cytoplasm by H(2)O(2), p40(phox) can acquire PI(3)P binding on targeted membranes in a p47(phox)-dependent manner and functions both as a "carrier" of the cytoplasmic Phox complex to phagosomes and an "adaptor" of oxidase assembly on phagosomes in cooperation with p47(phox), using positive feedback mechanisms.
- Published
- 2011
- Full Text
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171. Hepatitis C virus (HCV) proteins induce NADPH oxidase 4 expression in a transforming growth factor beta-dependent manner: a new contributor to HCV-induced oxidative stress.
- Author
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Boudreau HE, Emerson SU, Korzeniowska A, Jendrysik MA, and Leto TL
- Subjects
- Animals, Gene Expression Regulation, Enzymologic, Hep G2 Cells, Humans, Mice, NADPH Oxidase 4, NADPH Oxidases physiology, Reactive Oxygen Species metabolism, Superoxides metabolism, Hepacivirus physiology, NADPH Oxidases genetics, Oxidative Stress, Transforming Growth Factor beta physiology, Viral Proteins physiology
- Abstract
Viral hepatitis-induced oxidative stress accompanied by increased levels of transforming growth factor beta (TGF-beta) and hepatic fibrosis are hallmarks of hepatitis C virus (HCV) infection. The mechanisms of redox regulation in the pathogenesis of HCV-induced liver disease are not clearly understood. The results of our current studies suggest that reactive oxygen species (ROS) derived from Nox4, a member of the NADPH oxidase (Nox) family, could play a role in HCV-induced liver disease. We found that the expression of HCV (genotype 1a) cDNA constructs (full-length and subgenomic), core protein alone, viral RNA, or replicating HCV (JFH-AM2) induced Nox4 mRNA expression and ROS generation in human hepatocyte cell lines (Huh-7, Huh-7.5, HepG2, and CHL). Conversely, hepatocytes expressing Nox4 short hairpin RNA (shRNA) or an inactive dominant negative form of Nox4 showed decreased ROS production when cells were transfected with HCV. The promoters of both human and murine Nox4 were used to demonstrate transcriptional regulation of Nox4 mRNA by HCV, and a luciferase reporter tied to an approximately 2-kb promoter region of Nox4 identified HCV-responsive regulatory regions modulating the expression of Nox4. Furthermore, the human Nox4 promoter was responsive to TGF-beta1, and the HCV core-dependent induction of Nox4 was blocked by antibody against TGF-beta or the expression of dominant negative TGF-beta receptor type II. These findings identified HCV as a regulator of Nox4 gene expression and subsequent ROS production through an autocrine TGF-beta-dependent mechanism. Collectively, these data provide evidence that HCV-induced Nox4 contributes to ROS production and may be related to HCV-induced liver disease.
- Published
- 2009
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172. Redox warfare between airway epithelial cells and Pseudomonas: dual oxidase versus pyocyanin.
- Author
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Rada B and Leto TL
- Subjects
- Animals, Antioxidants metabolism, Epithelial Cells immunology, Epithelial Cells metabolism, Epithelial Cells microbiology, Humans, Lactoperoxidase immunology, NADP immunology, NADP metabolism, NADPH Oxidases immunology, Oxidation-Reduction, Oxidative Stress immunology, Pseudomonas Infections microbiology, Pseudomonas aeruginosa physiology, Pyocyanine immunology, Reactive Oxygen Species immunology, Respiratory Mucosa metabolism, Respiratory Mucosa microbiology, Lactoperoxidase metabolism, NADPH Oxidases metabolism, Pseudomonas Infections immunology, Pyocyanine metabolism, Reactive Oxygen Species metabolism, Respiratory Mucosa immunology
- Abstract
The importance of reactive oxygen species-dependent microbial killing by the phagocytic cell NADPH oxidase has been appreciated for some time, although only recently has an appreciation developed for the partnership of lactoperoxidase with related dual oxidases (Duox) within secretions of the airway surface layer. This system produces mild oxidants designed for extracellular killing that are effective against several airway pathogens, including Staphylococcus aureus, Burkholderia cepacia, and Pseudomonas aeruginosa. Establishment of chronic pseudomonas infections involves adaptations to resist oxidant-dependent killing by expression of a redox-active virulence factor, pyocyanin, that competitively inhibits epithelial Duox activity by consuming intracellular NADPH and producing superoxide, thereby inflicting oxidative stress on the host.
- Published
- 2009
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173. Cytosolic phospholipase A2alpha is targeted to the p47phox-PX domain of the assembled NADPH oxidase via a novel binding site in its C2 domain.
- Author
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Shmelzer Z, Karter M, Eisenstein M, Leto TL, Hadad N, Ben-Menahem D, Gitler D, Banani S, Wolach B, Rotem M, and Levy R
- Subjects
- Binding Sites, Cell Line, Tumor, Cell Membrane enzymology, Cytosol metabolism, Group IV Phospholipases A2 chemistry, Group IV Phospholipases A2 genetics, Humans, Models, Molecular, NADPH Oxidases chemistry, NADPH Oxidases deficiency, NADPH Oxidases genetics, Protein Binding, Protein Structure, Quaternary, Protein Transport, Cytochrome b Group metabolism, Group IV Phospholipases A2 metabolism, NADPH Oxidases metabolism
- Abstract
We have previously demonstrated a physical interaction between cytosolic phospholipase A2alpha (cPLA2) and the assembled NADPH oxidase on plasma membranes following neutrophil stimulation. The aim of the present study was to define the exact binding sites between these two enzymes. Here we show, based on blot overlay experiments, Förster resonance energy transfer analysis and studies in neutrophils from patients with chronic granulomatous disease deficient in p67phox or p47phox, that cPLA2 specifically binds to p47phox and that p47phox is sufficient to anchor cPLA2 to the assembled oxidase on the plasma membranes upon stimulation. Blot overlay and affinity binding experiments using subfragments of cPLA2 and p47phox demonstrated that the cPLA2-C2 domain and the p47phox-PX domain interact to form a complex that is resistant to high salt. Computational docking was used to identify hydrophobic peptides within these two domains that inhibited the association between the two enzymes and NADPH oxidase activity in electro-permeabilized neutrophils. These results were used in new docking computations that produced an interaction model. Based on this model, cPLA2-C2 domain mutations were designed to explore its interaction p47phox in neutrophil lysates. The triple mutant F35A/M38A/L39A of the cPLA2-C2 domain caused a slight inhibition of the affinity binding to p47phox, whereas the single mutant I67A was highly effective. The double mutant M59A/H115A of the p47phox-PX domain caused a significant inhibition of the affinity binding to cPLA2. Thus, Ile67 of the cPLA2-C2 domain is identified as a critical, centrally positioned residue in a hydrophobic interaction in the p47phox-PX domain.
- Published
- 2008
- Full Text
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174. Mechanism of angiotensin II-induced superoxide production in cells reconstituted with angiotensin type 1 receptor and the components of NADPH oxidase.
- Author
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Choi H, Leto TL, Hunyady L, Catt KJ, Bae YS, and Rhee SG
- Subjects
- Adaptor Proteins, Signal Transducing, Adaptor Proteins, Vesicular Transport genetics, Adaptor Proteins, Vesicular Transport metabolism, Androstadienes pharmacology, Animals, CHO Cells, Cell Line, Chromones pharmacology, Cricetinae, Cricetulus, Gene Expression drug effects, Humans, Indoles pharmacology, Maleimides pharmacology, Membrane Glycoproteins genetics, Membrane Glycoproteins metabolism, Morpholines pharmacology, Mutation, NADPH Oxidase 1, NADPH Oxidase 2, NADPH Oxidases genetics, Phosphatidylinositol 3-Kinases metabolism, Phosphoinositide-3 Kinase Inhibitors, Phosphoproteins genetics, Phosphoproteins metabolism, Protein Kinase C antagonists & inhibitors, Protein Kinase C physiology, Pyrazoles pharmacology, Pyrimidines pharmacology, Quinazolines, Receptor, Angiotensin, Type 1 genetics, Reverse Transcriptase Polymerase Chain Reaction, Tyrphostins pharmacology, Wortmannin, Angiotensin II pharmacology, NADPH Oxidases metabolism, Receptor, Angiotensin, Type 1 metabolism, Superoxides metabolism
- Abstract
The mechanism of angiotensin II (Ang II)-induced superoxide production was investigated with HEK293 or Chinese hamster ovary cells reconstituted with the angiotensin type 1 receptor (AT(1)R) and NADPH oxidase (either Nox1 or Nox2) along with a pair of adaptor subunits (either NOXO1 with NOXA1 or p47(phox) with p67(phox)). Ang II enhanced the activity of both Nox1 and Nox2 supported by either adaptor pair, with more effective activation of Nox1 in the presence of NOXO1 and NOXA1 and of Nox2 in the presence of p47(phox) and p67(phox). Expression of several AT(1)R mutants showed that interaction of the receptor with G proteins but not that with beta-arrestin or with other proteins (Jak2, phospholipase C-gamma1, SH2 domain-containing phosphatase 2) that bind to the COOH-terminal region of AT(1)R, was necessary for Ang II-induced superoxide production. The effects of constitutively active alpha subunits of G proteins and of various pharmacological agents implicated signaling by a pathway comprising AT(1)R, Galpha(q/11), phospholipase C-beta, and protein kinase C as largely, but not exclusively, responsible for Ang II-induced activation of Nox1 and Nox2 in the reconstituted cells. A contribution of Galpha(12/13), phospholipase D, and phosphatidyl-inositol 3-kinase to Ang II-induced superoxide generation was also suggested, whereas Src and the epidermal growth factor receptor did not appear to participate in this effect of Ang II. In reconstituted cells stimulated with Ang II, Nox2 exhibited a more sensitive response than Nox1 to the perturbation of protein kinase C, phosphatidylinositol 3-kinase, or the small GTPase Rac1.
- Published
- 2008
- Full Text
- View/download PDF
175. Oxidative innate immune defenses by Nox/Duox family NADPH oxidases.
- Author
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Rada B and Leto TL
- Subjects
- Animals, Gastrointestinal Tract enzymology, Gastrointestinal Tract immunology, Gastrointestinal Tract microbiology, Gene Expression, Humans, Mouth enzymology, Mouth immunology, Mouth microbiology, Multigene Family, NADPH Oxidases chemistry, NADPH Oxidases genetics, NADPH Oxidases metabolism, Neutrophils enzymology, Neutrophils immunology, Neutrophils microbiology, Oxygen metabolism, Phagocytosis, Phagosomes enzymology, Respiratory System enzymology, Respiratory System immunology, Respiratory System microbiology, Immunity, Innate, NADPH Oxidases immunology, Reactive Oxygen Species immunology
- Abstract
The importance of reactive oxygen species (ROS) in innate immunity was first recognized in professional phagocytes undergoing a 'respiratory burst'upon activation. This robust oxygen consumption is related to a superoxide-generating enzyme, the phagocytic NADPH oxidase (Nox2-based or phox). The oxidase is essential for microbial killing, since patients lacking a functional oxidase suffer from enhanced susceptibility to microbial infections. ROS derived from superoxide attack bacteria in the isolated niche of the neutrophil phagosome. The oxidase is electrogenic, alters ion currents across membranes, induces apoptosis, regulates cytokine production, influences gene expression, and promotes formation of extracellular traps. Recently, new homologues of Nox2 were discovered establishing the Nox family of NADPH oxidases that encompasses seven members. Nox1 is highly expressed in the colon epithelium, and can be induced by LPS or IFN- gamma. Nox4 was implicated in innate immunity since LPS induces Nox4-dependent ROS generation. Duox1 and Duox2 localize to the apical plasma membrane of epithelial cells in major airways, salivary glands, and the gastrointestinal tract, and provide extracellular hydrogen peroxide to lactoperoxidase to produce antimicrobial hypothiocyanite ions. Th1 and Th2 cytokines regulate expression of dual oxidases in human airways and may thereby act in host defense or in proinflammatory responses.
- Published
- 2008
- Full Text
- View/download PDF
176. Novel sources of reactive oxygen species in the human body.
- Author
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Orient A, Donkó A, Szabó A, Leto TL, and Geiszt M
- Subjects
- Gene Expression Regulation, Enzymologic, Humans, Kidney enzymology, Phagocytes enzymology, Phagocytes physiology, Superoxides metabolism, NADPH Oxidases genetics, NADPH Oxidases metabolism, Reactive Oxygen Species metabolism
- Published
- 2007
- Full Text
- View/download PDF
177. A regulated adaptor function of p40phox: distinct p67phox membrane targeting by p40phox and by p47phox.
- Author
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Ueyama T, Tatsuno T, Kawasaki T, Tsujibe S, Shirai Y, Sumimoto H, Leto TL, and Saito N
- Subjects
- Animals, COS Cells, Chlorocebus aethiops, Humans, Mice, Mutation, Phagocytosis, Phagosomes chemistry, Phosphatidylinositol Phosphates metabolism, Phosphoproteins analysis, Phosphoproteins genetics, Protein Structure, Tertiary genetics, Protein Transport, Receptors, IgG immunology, Sequence Analysis, DNA, Cell Membrane immunology, NADPH Oxidases metabolism, Phagosomes immunology, Phosphoproteins metabolism
- Abstract
In the phagocytic cell, NADPH oxidase (Nox2) system, cytoplasmic regulators (p47(phox), p67(phox), p40(phox), and Rac) translocate and associate with the membrane-spanning flavocytochrome b(558), leading to activation of superoxide production. We examined membrane targeting of phox proteins and explored conformational changes in p40(phox) that regulate its translocation to membranes upon stimulation. GFP-p40(phox) translocates to early endosomes, whereas GFP-p47(phox) translocates to the plasma membrane in response to arachidonic acid. In contrast, GFP-p67(phox) does not translocate to membranes when expressed alone, but it is dependent on p40(phox) and p47(phox) for its translocation to early endosomes or the plasma membrane, respectively. Translocation of GFP-p40(phox) or GFP-p47(phox) to their respective membrane-targeting sites is abolished by mutations in their phox (PX) domains that disrupt their interactions with their cognate phospholipid ligands. Furthermore, GFP-p67(phox) translocation to either membrane is abolished by mutations that disrupt its interaction with p40(phox) or p47(phox). Finally, we detected a head-to-tail (PX-Phox and Bem1 [PB1] domain) intramolecular interaction within p40(phox) in its resting state by deletion mutagenesis, cell localization, and binding experiments, suggesting that its PX domain is inaccessible to interact with phosphatidylinositol 3-phosphate without cell stimulation. Thus, both p40(phox) and p47(phox) function as diverse p67(phox) "carrier proteins" regulated by the unmasking of membrane-targeting domains in distinct mechanisms.
- Published
- 2007
- Full Text
- View/download PDF
178. Subcellular localization and function of alternatively spliced Noxo1 isoforms.
- Author
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Ueyama T, Lekstrom K, Tsujibe S, Saito N, and Leto TL
- Subjects
- Adaptor Proteins, Signal Transducing, Alternative Splicing, Amino Acid Sequence, Animals, Base Sequence, Cell Line, Cell Membrane enzymology, Enzyme Activation physiology, Humans, Immunoblotting, Isoenzymes genetics, Isoenzymes metabolism, Isoenzymes ultrastructure, Microscopy, Confocal, Molecular Sequence Data, NADPH Oxidases genetics, NADPH Oxidases metabolism, NADPH Oxidases ultrastructure, Reverse Transcriptase Polymerase Chain Reaction, Transfection, Adaptor Proteins, Vesicular Transport genetics, Adaptor Proteins, Vesicular Transport metabolism, Adaptor Proteins, Vesicular Transport ultrastructure, Subcellular Fractions enzymology, Subcellular Fractions ultrastructure
- Abstract
Nox organizer 1 (Noxo1), a p47(phox) homolog, is produced as four isoforms with unique N-terminal PX domains derived by alternative mRNA splicing. We compared the subcellular distribution of these isoforms or their isolated PX domains produced as GFP fusion proteins, as well as their ability to support Nox1 activity in several transfected models. Noxo1alpha, beta, gamma, and delta show different subcellular localization patterns, determined by their PX domains. In HEK293 cells, Noxo1beta exhibits prominent plasma membrane binding, Noxo1gamma shows plasma membrane and nuclear associations, and Noxo1alpha and delta localize primarily on intracellular vesicles or cytoplasmic aggregates, but not the plasma membrane. Nox1 activity correlates with Noxo1 plasma membrane binding in HEK293 cells, since Noxo1beta supports the highest activity and Noxo1gamma and Noxo1alpha support moderate or low activities, respectively. In COS-7 cells, where Noxo1alpha localizes on the plasma membrane, the activities supported by the three isoforms (alpha, beta, and gamma) do not differ significantly. The PX domains of beta and gamma bind the same phospholipids, including phosphatidic acid. These results indicate that the variant PX domains are unique determinants of Noxo1 localization and Nox1 function. Finally, the overexpressed Noxo1 isoforms do not affect p22(phox) localization, although Nox1 is needed to transport p22(phox) to the plasma membrane.
- Published
- 2007
- Full Text
- View/download PDF
179. Two different pathways are involved in peroxynitrite-induced senescence and apoptosis of human erythrocytes.
- Author
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Pietraforte D, Matarrese P, Straface E, Gambardella L, Metere A, Scorza G, Leto TL, Malorni W, and Minetti M
- Subjects
- Carbon Dioxide metabolism, Flow Cytometry, Humans, Oxidation-Reduction drug effects, Apoptosis drug effects, Cellular Senescence drug effects, Erythrocytes drug effects, Peroxynitrous Acid metabolism
- Abstract
CO(2) changes the biochemistry of peroxynitrite basically in two ways: (i) nitrating species is the CO(3)(-) / ()NO(2) radical pair, and (ii) peroxynitrite diffusion distance is significantly reduced. For peroxynitrite generated extracellularly this last effect is particularly dramatic at low cell density because CO(3)(-) and ()NO(2) are short-lived and decay mostly in the extracellular space or at the cell surface/membrane. This study was aimed to distinguish between peroxynitrite-induced extra- and intracellular modifications of red blood cells (RBC). Our results show that at low cell density and in the presence of CO(2) peroxynitrite induced the oxidation of surface thiols, the formation of 3-nitrotyrosine and DMPO-RBC adducts, and the down-regulation of glycophorins A and C (biomarkers of senescence). Reactivation of glycolysis reversed only the oxidation of surface thiols. Without CO(2) peroxynitrite also induced the oxidation of hemoglobin and glutathione, the accumulation of lactate, a decrease in ATP, the clustering of band 3, the externalization of phosphatidylserine, and the activation of caspases 8 and 3 (biomarkers of apoptosis). The latter biomarkers were all reversed by reactivation of glycolysis. We hypothesize that cell senescence could (generally) be derived by irreversible radical-mediated oxidation of membrane targets, while the appearance of apoptotic biomarkers could be bolstered by oxidation of intracellular targets. These results suggest that, depending on extracellular homolysis or diffusion to the intracellular space, peroxynitrite prompts RBCs toward either senescence or apoptosis through different oxidation mechanisms.
- Published
- 2007
- Full Text
- View/download PDF
180. The K-562 cell model for analysis of neutrophil NADPH oxidase function.
- Author
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Leto TL, Lavigne MC, Homoyounpour N, Lekstrom K, Linton G, Malech HL, and de Mendez I
- Subjects
- Blotting, Western, Cell Fractionation, Cell Membrane metabolism, Humans, NADPH Oxidases genetics, NADPH Oxidases metabolism, Plasmids, Protein Transport, Transfection methods, K562 Cells, Models, Biological, NADPH Oxidases physiology, Neutrophils enzymology
- Abstract
Polymorphonuclear neutrophils (PMN) have a remarkable capacity for generation of large amounts of reactive oxygen species in response to a variety of infectious or inflammatory stimuli, a process known as the respiratory burst that involves activation of a multicomponent NADPH oxidase. Given their short life span, PMN are not amenable to most molecular biology methods for studying activation of this oxidant-generating system. We have explored a variety of methods for introduction of components of the phagocytic oxidase (phox system) into the promyelocytic erythroleukemia cell line, K-562. Here, we describe a series of cloned K-562 cell lines that were retrovirally transduced for stable production of one or more essential components of the phagocytic oxidase (phox) complex. We outline methods for the use of these transfectable cells for investigating structure, function, and signaling requirements for assembly and activation of the phox system. These versatile lines can be used to examine effects of genetic polymorphisms or mutations in phox components associated with chronic granulomatous disease, to serve as a system for testing gene therapy vectors designed to correct the defective oxidase, to study cross-functioning with recently described phox component homologs, or to explore signaling components involved in regulation of the respiratory burst.
- Published
- 2007
- Full Text
- View/download PDF
181. The Nox family of NAD(P)H oxidases: host defense and beyond.
- Author
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Geiszt M and Leto TL
- Subjects
- Animals, Humans, Models, Biological, NADH, NADPH Oxidoreductases chemistry, NADH, NADPH Oxidoreductases genetics, NADPH Oxidases, Reactive Oxygen Species metabolism, NADH, NADPH Oxidoreductases physiology
- Published
- 2004
- Full Text
- View/download PDF
182. Analysis of mRNA transcripts from the NAD(P)H oxidase 1 (Nox1) gene. Evidence against production of the NADPH oxidase homolog-1 short (NOH-1S) transcript variant.
- Author
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Geiszt M, Lekstrom K, and Leto TL
- Subjects
- Alternative Splicing, Animals, Base Sequence, Blotting, Northern, COS Cells, Cell Line, Cell Line, Tumor, Colon metabolism, DNA, Complementary metabolism, Databases as Topic, Exons, Genetic Variation, Humans, Models, Genetic, Molecular Sequence Data, Myoblasts metabolism, NADPH Oxidase 1, NADPH Oxidases chemistry, NADPH Oxidases metabolism, Protons, RNA, Messenger metabolism, Repetitive Sequences, Nucleic Acid, Reverse Transcriptase Polymerase Chain Reaction, Ribonucleases chemistry, Ribonucleases metabolism, Superoxides chemistry, Time Factors, Transcription, Genetic, Transfection, NADPH Oxidases genetics
- Abstract
Recent reports indicate that NAD(P)H oxidase 1 (Nox1) mRNA undergoes alternative splicing, producing a short transcript (NOH-1S) encoding a novel H+ channel. Although the H+ transport properties of NOH-1S-transfected cells resemble those of many cells, the production of a NOH-1S protein was never documented. We characterized Nox1 transcripts in colon-derived cells and present evidence that mRNA splicing does not produce NOH-1S; rather, NOH-1S appears to be an artifact of template switching during cDNA synthesis. The NOH-1S transcript was not observed by Northern blotting, despite claims of its abundance based on RNase protection assays. The shortened cDNA was generated by avian myeloblastosis virus reverse transcriptase, but not by thermally stable reverse transcriptase under conditions that produce full-length Nox1. Analysis of shortened cDNAs detected NOH-1S sequence and other variants that differ at the alleged splice junction site. Although no appropriate RNA splicing sites were found within Nox1 to account for NOH-1S formation, we found repetitive sequence elements bordering the deleted region, which could promote intramolecular template switching during cDNA synthesis. Template switching was confirmed in vitro, where the deleted cDNA was generated by avian myeloblastosis virus reverse transcriptase from a synthetic, full-length Nox1 RNA template. A survey of the expressed sequence tags database suggests that similar switching phenomena occur between repetitive elements in other Nox family transcripts, indicating such cloning artifacts are common. In contrast, genuine RNA splicing does account for another Nox1 transcript lacking the entire exon 11, which is abundant in colon cells but encodes a protein incapable of supporting superoxide production.
- Published
- 2004
- Full Text
- View/download PDF
183. Characterization of a dipeptide motif regulating IFN-gamma receptor 2 plasma membrane accumulation and IFN-gamma responsiveness.
- Author
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Rosenzweig SD, Schwartz OM, Brown MR, Leto TL, and Holland SM
- Subjects
- Amino Acid Motifs genetics, Amino Acid Motifs immunology, Cell Aggregation genetics, Cell Aggregation immunology, Cell Line, Cell Line, Transformed, Cell Membrane genetics, Dipeptides genetics, Down-Regulation genetics, Genetic Vectors, Green Fluorescent Proteins, Humans, Interferon-gamma antagonists & inhibitors, Interferon-gamma metabolism, Intracellular Fluid chemistry, Intracellular Fluid immunology, Intracellular Fluid metabolism, Luminescent Proteins genetics, Mannosidases metabolism, Mannosidases pharmacology, Mutagenesis, Site-Directed, Protein Sorting Signals genetics, Receptors, Interferon genetics, Receptors, Interferon physiology, Recombinant Fusion Proteins biosynthesis, Recombinant Fusion Proteins metabolism, Recombinant Fusion Proteins physiology, Signal Transduction genetics, Signal Transduction immunology, Transfection, Cell Membrane immunology, Cell Membrane metabolism, Dipeptides chemistry, Dipeptides physiology, Interferon-gamma physiology, Receptors, Interferon metabolism
- Abstract
The IFN-gammaR complex is composed of two IFN-gammaR1 and two IFN-gammaR2 polypeptide chains. Although IFN-gammaR1 is constitutively expressed on all nucleated cells, IFN-gammaR2 membrane display is selective and tightly regulated. We created a series of fluorescent-tagged IFN-gammaR2 expression constructs to follow the molecule's cell surface expression and intracellular distribution. Truncation of the receptor immediately upstream of Leu-Ile 255-256 (254X) created a receptor devoid of signaling that overaccumulated on the cell surface. In addition, this truncated receptor inhibited wild-type IFN-gammaR2 activity and therefore exerted a dominant negative effect. In-frame deletion (255Delta2) or alanine substitution (LI255-256AA) of these amino acids created mutants that overaccumulated on the plasma membrane, but had enhanced function. Single amino acid substitutions (L255A or I256A) had a more modest effect. In-frame deletions upstream (253Delta2), but not downstream (257Delta2), of Leu-Ile 255-256 also led to overaccumulation. A truncation within the IFN-gammaR2 Jak2 binding site (270X) led to a mutant devoid of function that did not overaccumulate and did not affect wild-type IFN-gammaR2 signaling. We have created a series of novel mutants of IFN-gammaR2 that have facilitated the identification of intracellular domains that control IFN-gammaR2 accumulation and IFN-gamma responsiveness. In contrast to IFN-gammaR1, not only dominant negative, but also dominant gain-of-function, mutations were created through manipulation of IFN-gammaR2 Leu-Ile 255-256. These IFN-gammaR2 mutants will allow fine dissection of the role of IFN-gamma signaling in immunity., (Copyright 2004 The American Association of Immunologists, Inc.)
- Published
- 2004
- Full Text
- View/download PDF
184. Unique targeting of cytosolic phospholipase A2 to plasma membranes mediated by the NADPH oxidase in phagocytes.
- Author
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Shmelzer Z, Haddad N, Admon E, Pessach I, Leto TL, Eitan-Hazan Z, Hershfinkel M, and Levy R
- Subjects
- Humans, Phospholipases A2, Precipitin Tests, Cell Membrane metabolism, Cytoplasm metabolism, NADPH Oxidases metabolism, Phagocytes metabolism, Phospholipases A metabolism
- Abstract
Cytosolic phospholipase A2 (cPLA2)-generated arachidonic acid (AA) has been shown to be an essential requirement for the activation of NADPH oxidase, in addition to its being the major enzyme involved in the formation of eicosanoid at the nuclear membranes. The mechanism by which cPLA2 regulates NADPH oxidase activity is not known, particularly since the NADPH oxidase complex is localized in the plasma membranes of stimulated cells. The present study is the first to demonstrate that upon stimulation cPLA2 is transiently recruited to the plasma membranes by a functional NADPH oxidase in neutrophils and in granulocyte-like PLB-985 cells. Coimmunoprecipitation experiments and double labeling immunofluorescence analysis demonstrated the unique colocalization of cPLA2 and the NADPH oxidase in plasma membranes of stimulated cells, in correlation with the kinetic burst of superoxide production. A specific affinity in vitro binding was detected between GST-p47phox or GST-p67phox and cPLA2 in lysates of stimulated cells. The association between these two enzymes provides the molecular basis for AA released by cPLA2 to activate the assembled NADPH oxidase. The ability of cPLA2 to regulate two different functions in the same cells (superoxide generation and eicosanoid production) is achieved by a novel dual subcellular localization of cPLA2 to different targets.
- Published
- 2003
- Full Text
- View/download PDF
185. Proteins homologous to p47phox and p67phox support superoxide production by NAD(P)H oxidase 1 in colon epithelial cells.
- Author
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Geiszt M, Lekstrom K, Witta J, and Leto TL
- Subjects
- Amino Acid Sequence, Animals, Colon cytology, Colon enzymology, DNA-Binding Proteins chemistry, DNA-Binding Proteins genetics, Epithelial Cells enzymology, Epithelial Cells metabolism, Genes, Tumor Suppressor, In Situ Hybridization, Mice, Molecular Sequence Data, Phosphoproteins genetics, RNA, Messenger genetics, Sequence Homology, Amino Acid, Transcription Factors, Tumor Suppressor Proteins, Colon metabolism, DNA-Binding Proteins physiology, NADPH Oxidases metabolism, Phosphoproteins chemistry, Phosphoproteins physiology, Superoxides metabolism, Trans-Activators
- Abstract
Superoxide production by phagocytes involves activation of a multi-component NADPH oxidase. Recently, several homologues of the catalytic component of the phagocyte oxidase, gp91phox, were identified in various tissues. Here we describe two proteins, p41 and p51, with significant homology to two cytosolic components of the phagocytic oxidase, p47phox and p67phox. Like p47phox, p41 contains an amino-terminal Phox homology domain, two SH3 domains, and a conserved carboxyl-terminal, proline-rich motif. Similarly, p51 is homologous to p67phox, containing four amino-terminal tetratrico-peptide repeats, a conserved "activation domain" motif, a PB1 domain, and a carboxyl-terminal SH3 domain. The highest levels of p41 transcript are detected in the colon and in other gastrointestinal tissues that express Nox1, the predominant gp91phox homologue in these tissues. In contrast, the p51 transcript showed a more widespread expression pattern, suggesting that it may support other tissue-specific oxidases. Mouse colon in situ hybridization detected both transcripts in the epithelial cells of colon crypts. Heterologous co-expression of p41 and p51 significantly enhances the superoxide-generating activity of Nox1-expressing cells; thus, p41 and p51 appear to be novel regulators of Nox1. These proteins also support the activity of gp91phox, albeit at much lower levels than the cytosolic phox counterparts. Our results suggest colon epithelial cells contain a multi-component NAD(P)H oxidase with a molecular architecture similar to the phagocytic oxidase.
- Published
- 2003
- Full Text
- View/download PDF
186. Identification of Renox, a Novel Superoxide Producing Enzyme in Kidney.
- Author
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Geiszt M and Leto TL
- Published
- 2001
- Full Text
- View/download PDF
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