ABSTRACT
Oxidative stress is two sided: Whereas excessive oxidant challenge causes damage to biomolecules, maintenance of a physiological level of oxidant challenge, termed oxidative eustress, is essential for governing life processes through redox signaling. Recent interest has focused on the intricate ways by which redox signaling integrates these converse properties. Redox balance is maintained by prevention, interception, and repair, and concomitantly the regulatory potential of molecular thiol-driven master switches such as Nrf2/Keap1 or NF-κB/IκB is used for system-wide oxidative stress response. Nonradical species such as hydrogen peroxide (H2O2) or singlet molecular oxygen, rather than free-radical species, perform major second messenger functions. Chemokine-controlled NADPH oxidases and metabolically controlled mitochondrial sources of H2O2 as well as glutathione- and thioredoxin-related pathways, with powerful enzymatic back-up systems, are responsible for fine-tuning physiological redox signaling. This makes for a rich research field spanning from biochemistry and cell biology into nutritional sciences, environmental medicine, and molecular knowledge-based redox medicine.
Subject(s)
Kelch-Like ECH-Associated Protein 1/metabolism , Mitochondria/metabolism , NADPH Oxidases/metabolism , NF-E2-Related Factor 2/metabolism , NF-kappa B/metabolism , Oxidative Stress , Gene Expression Regulation , Glutathione/metabolism , Humans , Hydrogen Peroxide/metabolism , Kelch-Like ECH-Associated Protein 1/genetics , NADPH Oxidases/genetics , NF-E2-Related Factor 2/genetics , NF-KappaB Inhibitor alpha/genetics , NF-KappaB Inhibitor alpha/metabolism , NF-kappa B/genetics , Oxidation-Reduction , Signal Transduction , Singlet Oxygen/metabolism , Thioredoxins/genetics , Thioredoxins/metabolismABSTRACT
Neutrophils are essential for killing bacteria and other microorganisms, and they also have a significant role in regulating the inflammatory response. Stimulated neutrophils activate their NADPH oxidase (NOX2) to generate large amounts of superoxide, which acts as a precursor of hydrogen peroxide and other reactive oxygen species that are generated by their heme enzyme myeloperoxidase. When neutrophils engulf bacteria they enclose them in small vesicles (phagosomes) into which superoxide is released by activated NOX2 on the internalized neutrophil membrane. The superoxide dismutates to hydrogen peroxide, which is used by myeloperoxidase to generate other oxidants, including the highly microbicidal species hypochlorous acid. NOX activation occurs at other sites in the cell, where it is considered to have a regulatory function. Neutrophils also release oxidants, which can modify extracellular targets and affect the function of neighboring cells. We discuss the identity and chemical properties of the specific oxidants produced by neutrophils in different situations, and what is known about oxidative mechanisms of microbial killing, inflammatory tissue damage, and signaling.
Subject(s)
Chloramines/metabolism , Hydrogen Peroxide/metabolism , Hypochlorous Acid/metabolism , Neutrophils/immunology , Superoxides/metabolism , Thiocyanates/metabolism , Cell Membrane/drug effects , Cells, Cultured , Chloramines/immunology , Gene Expression , Humans , Hydrogen Peroxide/immunology , Hypochlorous Acid/immunology , Membrane Glycoproteins/agonists , Membrane Glycoproteins/genetics , Membrane Glycoproteins/immunology , NADPH Oxidase 2 , NADPH Oxidases/genetics , NADPH Oxidases/immunology , Neutrophils/cytology , Neutrophils/drug effects , Oxidation-Reduction , Peroxidase/genetics , Peroxidase/immunology , Signal Transduction , Superoxides/immunology , Tetradecanoylphorbol Acetate/pharmacology , Thiocyanates/immunology , Zymosan/pharmacologyABSTRACT
The ability to continuously adjust posture and balance is necessary for reliable motor behavior. Vestibular and proprioceptive systems influence postural adjustments during movement by signaling functionally complementary sensory information. Using viral tracing and mouse genetics, we reveal two patterns of synaptic specificity between brainstem vestibular neurons and spinal motor neurons, established through distinct mechanisms. First, vestibular input targets preferentially extensor over flexor motor pools, a pattern established by developmental refinement in part controlled by vestibular signaling. Second, vestibular input targets slow-twitch over fast motor neuron subtypes within extensor pools, while proprioceptors exhibit inversely correlated connectivity profiles. Genetic manipulations affecting the functionality of proprioceptive feedback circuits lead to adjustments in vestibular input to motor neuron subtypes counterbalancing the imposed changes, without changing the sparse vestibular input to flexor pools. Thus, two sensory signaling systems interact to establish complementary synaptic input patterns to the final site of motor output processing.
Subject(s)
Postural Balance , Posture , Proprioception , Vestibular Nuclei/metabolism , Animals , Mice , Mice, 129 Strain , Mice, Inbred C57BL , Motor Neurons/metabolism , NADPH Oxidases/genetics , NADPH Oxidases/metabolism , Synapses , Vestibule, Labyrinth/metabolismABSTRACT
The precise localization of extracellular matrix and cell wall components is of critical importance for multicellular organisms. Lignin is a major cell wall modification that often forms intricate subcellular patterns that are central to cellular function. Yet the mechanisms of lignin polymerization and the subcellular precision of its formation remain enigmatic. Here, we show that the Casparian strip, a lignin-based, paracellular diffusion barrier in plants, forms as a precise, median ring by the concerted action of a specific, localized NADPH oxidase, brought into proximity of localized peroxidases through the action of Casparian strip domain proteins (CASPs). Our findings in Arabidopsis provide a simple mechanistic model of how plant cells regulate lignin formation with subcellular precision. We speculate that scaffolding of NADPH oxidases to the downstream targets of the reactive oxygen species (ROS) that they produce might be a widespread mechanism to ensure specificity and subcellular precision of ROS action within the extracellular matrix.
Subject(s)
Arabidopsis/cytology , Arabidopsis/enzymology , Lignin/metabolism , NADPH Oxidases/metabolism , Arabidopsis/chemistry , Arabidopsis/metabolism , Biological Transport , Cell Wall/metabolism , Diffusion , Hydrogen Peroxide/metabolism , NADPH Oxidases/genetics , Plant Proteins/metabolism , Polymerization , Superoxides/metabolismABSTRACT
A growing wealth of data suggest that reactive oxygen species (ROS) signalling might be crucial in conferring embryonic or adult stem cells their specific properties. However, how stem cells control ROS production and scavenging, and how ROS in turn contribute to stemness, remain poorly understood. Using the Xenopus retina as a model system, we first investigated the redox status of retinal stem cells (RSCs). We discovered that they exhibit higher ROS levels compared with progenitors and retinal neurons, and express a set of specific redox genes. We next addressed the question of ROS functional involvement in these cells. Using pharmacological or genetic tools, we demonstrate that inhibition of NADPH oxidase-dependent ROS production increases the proportion of quiescent RSCs. Surprisingly, this is accompanied by an apparent acceleration of the mean division speed within the remaining proliferating pool. Our data further unveil that such impact on RSC cell cycling is achieved by modulation of the Wnt/Hedgehog signalling balance. Altogether, we highlight that RSCs exhibit distinctive redox characteristics and exploit NADPH oxidase signalling to limit quiescence and fine-tune their proliferation rate.
Subject(s)
Adult Stem Cells , Neural Stem Cells , Animals , Xenopus laevis/metabolism , Reactive Oxygen Species , Cell Proliferation , Hedgehog Proteins , Retina/metabolism , Adult Stem Cells/metabolism , NADPH Oxidases/genetics , Wnt Signaling PathwayABSTRACT
Plants produce a burst of reactive oxygen species (ROS) after pathogen infection to successfully activate immune responses. During pattern-triggered immunity (PTI), ROS are primarily generated by the NADPH oxidase RESPIRATORY BURST OXIDASE HOMOLOG D (RBOHD). RBOHD is degraded in the resting state to avoid inappropriate ROS production; however, the enzyme mediating RBOHD degradation and how to prevent RBOHD degradation after pathogen infection is unclear. In this study, we identified an Arabidopsis (Arabidopsis thaliana) vacuole-localized papain-like cysteine protease, XYLEM CYSTEINE PEPTIDASE 1 (XCP1), and its inhibitor CYSTATIN 6 (CYS6). Pathogen-associated molecular pattern-induced ROS burst and resistance were enhanced in the xcp1 mutant but were compromised in the cys6 mutant, indicating that XCP1 and CYS6 oppositely regulate PTI responses. Genetic and biochemical analyses revealed that CYS6 interacts with XCP1 and depends on XCP1 to enhance PTI. Further experiments showed that XCP1 interacts with RBOHD and accelerates RBOHD degradation in a vacuole-mediated manner. CYS6 inhibited the protease activity of XCP1 toward RBOHD, which is critical for RBOHD accumulation upon pathogen infection. As CYS6, XCP1, and RBOHD are conserved in all plant species tested, our findings suggest the existence of a conserved strategy to precisely regulate ROS production under different conditions by modulating the stability of RBOHD.
Subject(s)
Arabidopsis Proteins , Arabidopsis , Cysteine Proteases , Arabidopsis Proteins/metabolism , Cysteine/metabolism , Reactive Oxygen Species/metabolism , Cystatin M/metabolism , Innate Immunity Recognition , Arabidopsis/metabolism , NADPH Oxidases/genetics , NADPH Oxidases/metabolism , Cysteine Proteases/metabolism , Plant Immunity/geneticsABSTRACT
Commensal microbes colonize the gut epithelia of virtually all animals and provide several benefits to their hosts. Changes in commensal populations can lead to dysbiosis, which is associated with numerous pathologies and decreased lifespan. Peptidoglycan recognition proteins (PGRPs) are important regulators of the commensal microbiota and intestinal homeostasis. Here, we found that a null mutation in Drosophila PGRP-SD was associated with overgrowth of Lactobacillus plantarum in the fly gut and a shortened lifespan. L. plantarum-derived lactic acid triggered the activation of the intestinal NADPH oxidase Nox and the generation of reactive oxygen species (ROS). In turn, ROS production promoted intestinal damage, increased proliferation of intestinal stem cells, and dysplasia. Nox-mediated ROS production required lactate oxidation by the host intestinal lactate dehydrogenase, revealing a host-commensal metabolic crosstalk that is probably broadly conserved. Our findings outline a mechanism whereby host immune dysfunction leads to commensal dysbiosis that in turn promotes age-related pathologies.
Subject(s)
Drosophila/physiology , Lactic Acid/metabolism , Longevity , Microbiota , NADPH Oxidases/metabolism , Reactive Oxygen Species/metabolism , Animals , Carrier Proteins/genetics , Carrier Proteins/metabolism , Dysbiosis , Gene Expression , Intestinal Mucosa/immunology , Intestinal Mucosa/metabolism , Intestinal Mucosa/microbiology , L-Lactate Dehydrogenase/genetics , L-Lactate Dehydrogenase/metabolism , Mutation , NADPH Oxidases/genetics , Signal Transduction , SymbiosisABSTRACT
Essential for reactive oxygen species (EROS) protein is a recently identified molecular chaperone of NOX2 (gp91phox), the catalytic subunit of phagocyte NADPH oxidase. Deficiency in EROS is a recently identified cause for chronic granulomatous disease, a genetic disorder with recurrent bacterial and fungal infections. Here, we report a cryo-EM structure of the EROS-NOX2-p22phox heterotrimeric complex at an overall resolution of 3.56Å. EROS and p22phox are situated on the opposite sides of NOX2, and there is no direct contact between them. EROS associates with NOX2 through two antiparallel transmembrane (TM) α-helices and multiple ß-strands that form hydrogen bonds with the cytoplasmic domain of NOX2. EROS binding induces a 79° upward bend of TM2 and a 48° backward rotation of the lower part of TM6 in NOX2, resulting in an increase in the distance between the two hemes and a shift of the binding site for flavin adenine dinucleotide (FAD). These conformational changes are expected to compromise superoxide production by NOX2, suggesting that the EROS-bound NOX2 is in a protected state against activation. Phorbol myristate acetate, an activator of NOX2 in vitro, is able to induce dissociation of NOX2 from EROS with concurrent increase in FAD binding and superoxide production in a transfected COS-7 model. In differentiated neutrophil-like HL-60, the majority of NOX2 on the cell surface is dissociated with EROS. Further studies are required to delineate how EROS dissociates from NOX2 during its transport to cell surface, which may be a potential mechanism for regulation of NOX2 activation.
Subject(s)
Cryoelectron Microscopy , NADPH Oxidase 2 , NADPH Oxidases , Phagocytes , Humans , NADPH Oxidase 2/metabolism , NADPH Oxidase 2/genetics , NADPH Oxidase 2/chemistry , Phagocytes/metabolism , NADPH Oxidases/metabolism , NADPH Oxidases/genetics , NADPH Oxidases/chemistry , Protein Binding , Binding Sites , Granulomatous Disease, Chronic/metabolism , Granulomatous Disease, Chronic/genetics , Models, Molecular , Reactive Oxygen Species/metabolismABSTRACT
Chaperone-mediated autophagy (CMA) targets soluble proteins for lysosomal degradation. Here we found that CMA was activated in T cells in response to engagement of the T cell antigen receptor (TCR), which induced expression of the CMA-related lysosomal receptor LAMP-2A. In activated T cells, CMA targeted the ubiquitin ligase Itch and the calcineurin inhibitor RCAN1 for degradation to maintain activation-induced responses. Consequently, deletion of the gene encoding LAMP-2A in T cells caused deficient in vivo responses to immunization or infection with Listeria monocytogenes. Impaired CMA activity also occurred in T cells with age, which negatively affected their function. Restoration of LAMP-2A in T cells from old mice resulted in enhancement of activation-induced responses. Our findings define a role for CMA in regulating T cell activation through the targeted degradation of negative regulators of T cell activation.
Subject(s)
Autophagy/immunology , Lymphocyte Activation/immunology , Lysosomal-Associated Membrane Protein 2/immunology , Molecular Chaperones/immunology , Th1 Cells/immunology , Aging/immunology , Animals , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/biosynthesis , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/genetics , Calcineurin Inhibitors/metabolism , Calcium-Binding Proteins , Cells, Cultured , Dual Oxidases , Female , Humans , Immunization , Intracellular Signaling Peptides and Proteins/metabolism , Listeria monocytogenes/immunology , Listeriosis/immunology , Lysosomal-Associated Membrane Protein 2/biosynthesis , Lysosomal-Associated Membrane Protein 2/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Muscle Proteins/metabolism , NADPH Oxidases/genetics , Oxidative Stress/immunology , RNA Interference , RNA, Messenger/biosynthesis , RNA, Small Interfering , Reactive Oxygen Species/metabolism , Receptors, Antigen, T-Cell/immunology , Ubiquitin-Protein Ligases/metabolismABSTRACT
The actin cytoskeleton and reactive oxygen species (ROS) both play crucial roles in various cellular processes. Previous research indicated a direct interaction between two key components of these systems: the WAVE1 subunit of the WAVE regulatory complex (WRC), which promotes actin polymerization and the p47phox subunit of the NADPH oxidase 2 complex (NOX2), which produces ROS. Here, using carefully characterized recombinant proteins, we find that activated p47phox uses its dual Src homology 3 domains to bind to multiple regions within the WAVE1 and Abi2 subunits of the WRC, without altering WRC's activity in promoting Arp2/3-mediated actin polymerization. Notably, contrary to previous findings, p47phox uses the same binding pocket to interact with both the WRC and the p22phox subunit of NOX2, albeit in a mutually exclusive manner. This observation suggests that when activated, p47phox may separately participate in two distinct processes: assembling into NOX2 to promote ROS production and engaging with WRC to regulate the actin cytoskeleton.
Subject(s)
NADPH Oxidase 2 , Wiskott-Aldrich Syndrome Protein Family , Humans , Actin Cytoskeleton/genetics , Actin Cytoskeleton/metabolism , Actins/metabolism , NADPH Oxidase 2/metabolism , NADPH Oxidase 2/genetics , NADPH Oxidases/metabolism , NADPH Oxidases/genetics , Protein Binding , Reactive Oxygen Species/metabolism , Wiskott-Aldrich Syndrome Protein Family/metabolism , Wiskott-Aldrich Syndrome Protein Family/genetics , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Protein Subunits/genetics , Protein Subunits/metabolism , Actin-Related Protein 2-3 Complex/metabolism , Binding SitesABSTRACT
Salicylic acid (SA) is known to enhance salt tolerance in plants. However, the mechanism of SA-mediated response to high salinity in halophyte remains unclear. Using electrophysiological and molecular biological methods, we investigated the role of SA in response to high salinity in mangrove species, Kandelia obovata, a typical halophyte. Exposure of K. obovata roots to high salinity resulted in a rapid increase in endogenous SA produced by phenylalanine ammonia lyase pathway. The application of exogenous SA improved the salt tolerance of K. obovata, which depended on the NADPH oxidase-mediated H2O2. Exogenous SA and H2O2 increased Na+ efflux and reduced K+ loss by regulating the transcription levels of Na+ and K+ transport-related genes, thus reducing the Na+/K+ ratio in the salt-treated K. obovata roots. In addition, exogenous SA-enhanced antioxidant enzyme activity and its transcripts, and the expressions of four genes related to AsA-GSH cycle as well, then alleviated oxidative damages in the salt-treated K. obovata roots. However, the above effects of SA could be reversed by diphenyleneiodonium chloride (the NADPH oxidase inhibitor) and paclobutrazol (a SA biosynthesis inhibitor). Collectively, our results demonstrated that SA-induced salt tolerance of K. obovata depends on NADPH oxidase-generated H2O2 that affects Na+/K+ and redox homeostasis in response to high salinity.
Subject(s)
Homeostasis , Hydrogen Peroxide , NADPH Oxidases , Oxidation-Reduction , Plant Roots , Potassium , Salicylic Acid , Salt Tolerance , Sodium , Hydrogen Peroxide/metabolism , NADPH Oxidases/metabolism , NADPH Oxidases/genetics , Salicylic Acid/metabolism , Salicylic Acid/pharmacology , Potassium/metabolism , Salt Tolerance/genetics , Sodium/metabolism , Plant Roots/genetics , Plant Roots/physiology , Plant Roots/metabolism , Salt-Tolerant Plants/genetics , Salt-Tolerant Plants/metabolism , Salt-Tolerant Plants/physiology , Gene Expression Regulation, Plant , Rhizophoraceae/physiology , Rhizophoraceae/genetics , Rhizophoraceae/metabolism , Plant Proteins/genetics , Plant Proteins/metabolismABSTRACT
Root hair initiation is a highly regulated aspect of root development. The plant hormone ethylene and its precursor, 1-amino-cyclopropane-1-carboxylic acid, induce formation and elongation of root hairs. Using confocal microscopy paired with redox biosensors and dyes, we demonstrated that treatments that elevate ethylene levels lead to increased hydrogen peroxide accumulation in hair cells prior to root hair formation. In the ethylene-insensitive receptor mutant, etr1-3, and the signaling double mutant, ein3eil1, the increase in root hair number or reactive oxygen species (ROS) accumulation after ACC and ethylene treatment was lost. Conversely, etr1-7, a constitutive ethylene signaling receptor mutant, has increased root hair formation and ROS accumulation, similar to ethylene-treated Col-0 seedlings. The caprice and werewolf transcription factor mutants have decreased and elevated ROS levels, respectively, which are correlated with levels of root hair initiation. The rhd2-6 mutant, with a defect in the gene encoding the ROS-synthesizing RESPIRATORY BURST OXIDASE HOMOLOG C (RBOHC), and the prx44-2 mutant, which is defective in a class III peroxidase, showed impaired ethylene-dependent ROS synthesis and root hair formation via EIN3EIL1-dependent transcriptional regulation. Together, these results indicate that ethylene increases ROS accumulation through RBOHC and PRX44 to drive root hair formation.
Subject(s)
Arabidopsis Proteins , Arabidopsis , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Ethylenes/pharmacology , Gene Expression Regulation, Plant , Mutation/genetics , NADPH Oxidases/genetics , Plant Roots/metabolism , Reactive Oxygen Species/metabolismABSTRACT
The submergence-induced hypoxic condition negatively affects the plant growth and development, and causes early onset of senescence. Hypoxia alters the expression of a number of microRNAs (miRNAs). However, the molecular function of submergence stress-induced miRNAs in physiological or developmental changes and recovery remains poorly understood. Here, we show that miR775 is an Arabidopsis thaliana-specific young and unique miRNA that possibly evolved non-canonically. miR775 post-transcriptionally regulates GALACTOSYLTRANSFERASE 9 (GALT9) and their expression is inversely affected at 24â h of complete submergence stress. The overexpression of miR775 (miR775-Oe) confers enhanced recovery from submergence stress and reduced accumulation of RBOHD and ROS, in contrast to wild-type and MIM775 Arabidopsis shoot. A similar recovery phenotype in the galt9 mutant indicates the role of the miR775-GALT9 module in post-submergence recovery. We predicted that Golgi-localized GALT9 is potentially involved in protein glycosylation. The altered expression of senescence-associated genes (SAG12, SAG29 and ORE1), ethylene signalling (EIN2 and EIN3) and abscisic acid (ABA) biosynthesis (NCED3) pathway genes occurs in miR775-Oe, galt9 and MIM775 plants. Thus, our results indicate the role for the miR775-GALT9 module in post-submergence recovery through a crosstalk between the ethylene signalling and ABA biosynthesis pathways.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , Ethylenes/pharmacology , Galactosyltransferases/metabolism , MicroRNAs/metabolism , Plant Senescence/drug effects , Abscisic Acid/metabolism , Arabidopsis/genetics , Arabidopsis/growth & development , Arabidopsis Proteins/chemistry , Arabidopsis Proteins/genetics , Binding Sites , Cysteine Endopeptidases/genetics , Cysteine Endopeptidases/metabolism , Dioxygenases/genetics , Dioxygenases/metabolism , Galactosyltransferases/genetics , Golgi Apparatus/metabolism , MicroRNAs/chemistry , MicroRNAs/genetics , NADPH Oxidases/genetics , NADPH Oxidases/metabolism , Plant Leaves/genetics , Plant Leaves/metabolism , Plant Proteins/genetics , Plant Proteins/metabolism , Plants, Genetically Modified/genetics , Plants, Genetically Modified/growth & development , Plants, Genetically Modified/metabolism , Reactive Oxygen Species/metabolism , Receptors, Cell Surface/genetics , Receptors, Cell Surface/metabolism , Signal Transduction/genetics , Stress, PhysiologicalABSTRACT
The hydrogen peroxide (H2 O2 )-producing NADPH oxidase Nox4, forming a heterodimer with p22phox , is expressed in a variety of cells including those in the heart to mediate adaptive responses to cellular stresses such as hypoxia. Since Nox4 is constitutively active, H2 O2 production is controlled by its protein abundance. Hypoxia-induced Nox4 expression is observed in various types of cells and generally thought to be regulated at the transcriptional level. Here we show that hypoxia upregulates the Nox4 protein level and Nox4-catalyzed H2 O2 production without increasing the Nox4 mRNA in rat H9c2 cardiomyocytes. In these cells, the Nox4 protein is stabilized under hypoxic conditions in a manner dependent on the presence of p22phox . Cell treatment with the proteasome inhibitor MG132 results in a marked decrease of the Nox4 protein under both normoxic and hypoxic conditions, indicating that the proteasome pathway does not play a major role in Nox4 degradation. The decrease is partially restored by the autophagy inhibitor 3-methyladenine. Furthermore, the Nox4 protein level is upregulated by the lysosome inhibitors bafilomycin A1 and chloroquine. Thus, in cardiomyocytes, Nox4 appears to be degraded via an autophagy-related pathway, and its suppression by hypoxia likely stabilizes Nox4, leading to upregulation of Nox4-catalyzed H2 O2 production.
Subject(s)
Myocytes, Cardiac , Oxidoreductases , Rats , Animals , NADPH Oxidase 4/genetics , NADPH Oxidase 4/metabolism , Myocytes, Cardiac/metabolism , NADPH Oxidases/genetics , NADPH Oxidases/metabolism , Hypoxia , Autophagy , Reactive Oxygen Species/metabolismABSTRACT
X-linked chronic granulomatous disease (X-CGD) is a primary immunodeficiency caused by mutations in the CYBB gene, resulting in the inability of phagocytic cells to eliminate infections. To design a lentiviral vector (LV) capable of recapitulating the endogenous regulation and expression of CYBB, a bioinformatics-guided approach was used to elucidate the cognate enhancer elements regulating the native CYBB gene. Using this approach, we analyzed a 600-kilobase topologically associated domain of the CYBB gene and identified endogenous enhancer elements to supplement the CYBB promoter to develop MyeloVec, a physiologically regulated LV for the treatment of X-CGD. When compared with an LV currently in clinical trials for X-CGD, MyeloVec showed improved expression, superior gene transfer to hematopoietic stem and progenitor cells (HSPCs), corrected an X-CGD mouse model leading to complete protection against Burkholderia cepacia infection, and restored healthy donor levels of antimicrobial oxidase activity in neutrophils derived from HSPCs from patients with X-CGD. Our findings validate the bioinformatics-guided design approach and have yielded a novel LV with clinical promise for the treatment of X-CGD.
Subject(s)
Granulomatous Disease, Chronic , Animals , Mice , Granulomatous Disease, Chronic/genetics , Granulomatous Disease, Chronic/therapy , NADPH Oxidases/genetics , NADPH Oxidases/metabolism , NADPH Oxidase 2/genetics , Genetic Therapy/methods , MutationABSTRACT
Tissue wounding induces cutaneous sensory axon regeneration via hydrogen peroxide (H2O2) that is produced by the epithelial NADPH oxidase, Duox1. Sciatic nerve injury instead induces axon regeneration through neuronal uptake of the NADPH oxidase, Nox2, from macrophages. We therefore reasoned that the tissue environment in which axons are damaged stimulates distinct regenerative mechanisms. Here, we show that cutaneous axon regeneration induced by tissue wounding depends on both neuronal and keratinocyte-specific mechanisms involving H2O2 signaling. Genetic depletion of H2O2 in sensory neurons abolishes axon regeneration, whereas keratinocyte-specific H2O2 depletion promotes axonal repulsion, a phenotype mirrored in duox1 mutants. Intriguingly, cyba mutants, deficient in the essential Nox subunit, p22Phox, retain limited axon regenerative capacity but display delayed Wallerian degeneration and axonal fusion, observed so far only in invertebrates. We further show that keratinocyte-specific oxidation of the epidermal growth factor receptor (EGFR) at a conserved cysteine thiol (C797) serves as an attractive cue for regenerating axons, leading to EGFR-dependent localized epidermal matrix remodeling via the matrix-metalloproteinase, MMP-13. Therefore, wound-induced cutaneous axon de- and regeneration depend on the coordinated functions of NADPH oxidases mediating distinct processes following injury.
Subject(s)
Axons , Hydrogen Peroxide , NADPH Oxidases , Nerve Regeneration , Wound Healing , Zebrafish Proteins , Animals , Axons/physiology , Hydrogen Peroxide/metabolism , Keratinocytes/physiology , NADPH Oxidases/genetics , NADPH Oxidases/physiology , Nerve Regeneration/genetics , Sensory Receptor Cells/physiology , Wound Healing/genetics , Wound Healing/physiology , Zebrafish , Zebrafish Proteins/genetics , Zebrafish Proteins/physiologyABSTRACT
BACKGROUND: P47phox (neutrophil cytosolic factor-1) deficiency is the most common cause of autosomal recessive chronic granulomatous disease (CGD) and is considered to be associated with a milder clinical phenotype. Allogeneic hematopoietic cell transplantation (HCT) for p47phox CGD is not well-described. OBJECTIVES: We sought to study HCT for p47phox CGD in North America. METHODS: Thirty patients with p47phox CGD who received allogeneic HCT at Primary Immune Deficiency Treatment Consortium centers since 1995 were included. RESULTS: Residual oxidative activity was present in 66.7% of patients. In the year before HCT, there were 0.38 CGD-related infections per person-years. Inflammatory diseases, predominantly of the lungs and bowel, occurred in 36.7% of the patients. The median age at HCT was 9.1 years (range 1.5-23.6 years). Most HCTs (90%) were performed after using reduced intensity/toxicity conditioning. HCT sources were HLA-matched (40%) and -mismatched (10%) related donors or HLA-matched (36.7%) and -mismatched (13.3%) unrelated donors. CGD-related infections after HCT decreased significantly to 0.06 per person-years (P = .038). The frequency of inflammatory bowel disease and the use of steroids also decreased. The cumulative incidence of graft failure and second HCT was 17.9%. The 2-year overall and event-free survival were 92.3% and 82.1%, respectively, while at 5 years they were 85.7% and 77.0%, respectively. In the surviving patients evaluated, ≥95% donor myeloid chimerism at 1 and 2 years after HCT was 93.8% and 87.5%, respectively. CONCLUSIONS: Patients with p47phox CGD suffer from a significant disease burden that can be effectively alleviated by HCT. Similar to other forms of CGD, HCT should be considered for patients with p47phox CGD.
Subject(s)
Granulomatous Disease, Chronic , Hematopoietic Stem Cell Transplantation , NADPH Oxidases , Humans , Granulomatous Disease, Chronic/therapy , Granulomatous Disease, Chronic/genetics , NADPH Oxidases/genetics , Male , Female , Child , Child, Preschool , Adolescent , Infant , Young Adult , Transplantation, Homologous , Transplantation Conditioning/methods , Graft vs Host Disease , Adult , Treatment OutcomeABSTRACT
Stomatal defences are important for plants to prevent pathogen entry and further colonisation of leaves. Apoplastic reactive oxygen species (ROS) generated by NADPH oxidases and apoplastic peroxidases play an important role in activating stomatal closure upon perception of bacteria. However, downstream events, particularly the factors influencing cytosolic hydrogen peroxide (H2 O2 ) signatures in guard cells are poorly understood. We used the H2 O2 sensor roGFP2-Orp1 and a ROS-specific fluorescein probe to study intracellular oxidative events during stomatal immune response using Arabidopsis mutants involved in the apoplastic ROS burst. Surprisingly, the NADPH oxidase mutant rbohF showed over-oxidation of roGFP2-Orp1 by a pathogen-associated molecular pattern (PAMP) in guard cells. However, stomatal closure was not tightly correlated with high roGFP2-Orp1 oxidation. In contrast, RBOHF was necessary for PAMP-mediated ROS production measured by a fluorescein-based probe in guard cells. Unlike previous reports, the rbohF mutant, but not rbohD, was impaired in PAMP-triggered stomatal closure resulting in defects in stomatal defences against bacteria. Interestingly, RBOHF also participated in PAMP-induced apoplastic alkalinisation. The rbohF mutants were also partly impaired in H2 O2 -mediated stomatal closure at 100 µm while higher H2 O2 concentration up to 1 mm did not promote stomatal closure in wild-type plants. Our results provide novel insights on the interplay between apoplastic and cytosolic ROS dynamics and highlight the importance of RBOHF in plant immunity.
Subject(s)
Arabidopsis Proteins , Arabidopsis , NADPH Oxidases , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Fluoresceins , Hydrogen-Ion Concentration , Plant Stomata/physiology , Reactive Oxygen Species , NADPH Oxidases/genetics , NADPH Oxidases/metabolismABSTRACT
Exercise as a lifestyle modification is a frontline therapy for nonalcoholic fatty liver disease (NAFLD), but how components of exercise attenuate steatosis is unclear. To uncouple the effect of increased muscle mass from weight loss in obesity, myostatin knockout mice were bred on a lean and obese db/db background. Myostatin deletion increases gastrocnemius (Gastrocn.) mass and reduces hepatic steatosis and hepatic sterol regulatory element binding protein 1 (Srebp1) expression in obese mice, with no impact on adiposity or body weight. Interestingly, hypermuscularity reduces hepatic NADPH oxidase 1 (Nox1) expression but not NADPH oxidase 4 (Nox4) in db/db mice. To evaluate a deterministic function of Nox1 on steatosis, Nox1 knockout mice were bred on a lean and db/db background. NOX1 deletion significantly attenuates hepatic oxidant stress, steatosis, and Srebp1 programming in obese mice to parallel hypermuscularity, with no improvement in adiposity, glucose control, or hypertriglyceridemia to suggest off-target effects. Directly assessing the role of NOX1 on SREBP1, insulin (Ins)-mediated SREBP1 expression was significantly increased in either NOX1, NADPH oxidase organizer 1 (NOXO1), and NADPH oxidase activator 1 (NOXA1) or NOX5-transfected HepG2 cells versus ?-galactosidase control virus, indicating superoxide is the key mechanistic agent for the actions of NOX1 on SREBP1. Metabolic Nox1 regulators were evaluated using physiological, genetic, and diet-induced animal models that modulated upstream glucose and insulin signaling, identifying hyperinsulinemia as the key metabolic derangement explaining Nox1-induced steatosis in obesity. GEO data revealed that hepatic NOX1 predicts steatosis in obese humans with biopsy-proven NAFLD. Taken together, these data suggest that hypermuscularity attenuates Srebp1 expression in db/db mice through a NOX1-dependent mechanism.NEW & NOTEWORTHY This study documents a novel mechanism by which changes in body composition, notably increased muscle mass, protect against fatty liver disease. This mechanism involves NADPH oxidase 1 (NOX1), an enzyme that increases superoxide and increases insulin signaling, leading to increased fat accumulation in the liver. NOX1 may represent a new early target for preventing fatty liver to stave off later liver diseases such as cirrhosis or liver cancer.
Subject(s)
Non-alcoholic Fatty Liver Disease , Animals , Humans , Mice , Insulin/metabolism , Liver/metabolism , Mice, Knockout , Mice, Obese , Muscle, Skeletal/metabolism , Myostatin , NADPH Oxidase 1/metabolism , NADPH Oxidases/genetics , NADPH Oxidases/metabolism , Non-alcoholic Fatty Liver Disease/genetics , Obesity/metabolism , Superoxides/metabolismABSTRACT
Chronic granulomatous disease (CGD) primarily results from inherited defects in components of the nicotinamide adenine dinucleotide phosphate oxidase enzyme complex. These include gene defects in cytochrome B-245/558 subunit α/ß and neutrophil cytosolic factors 1, 2, and 4. Recently, homozygous loss-of-function variants in cytochrome B-245 chaperone 1 gene (CYBC1) have been discovered to cause CGD (CYBC1-CGD). Data on variant-proven CGD from low-income countries, the most underprivileged regions of the world, remain sparse due to numerous constraints. Herein, we report the first cohort of patients with CGD from Nepal, a low-income country in the Himalayas' challenging terrain. Our report includes a description of a new case of CYBC1 deficiency who was first diagnosed with CGD at our center. Only a dozen cases of CYBC1-CGD have been described in the literature thus far which have been reviewed comprehensively herein. Most of these patients have had significant infections and autoimmune/inflammatory manifestations. Pulmonary and invasive/disseminated bacterial/fungal infections were the most common followed by skin and soft-tissue infections. Inflammatory bowel disease (IBD) was the most common inflammatory manifestation (median age at diagnosis: 9 years) followed by episodes of recurrent/prolonged fever. Other autoimmune/inflammatory manifestations reported in CYBC1-CGD include acute pancreatitis, hemophagocytic lymphohistiocytosis, systemic granulomatosis, interstitial lung disease, arthritis, autoimmune hemolytic anemia, uveitis, nephritis, and eczema. Our analysis shows that patients with CYBC1-CGD are at a significantly higher risk of IBD-like illness as compared to other forms of CGD which merits further confirmatory studies in the future.