Hydrogen peroxide (H
2O
2) has important messenger and effector functions in the plant and animal kingdom. Phagocytes produce H
2O
2 to kill pathogens, and epithelial cells of large airways have also ...been reported to produce H
2O
2 for signaling and host defense purposes. In this report, we show for the first time that urothelial cells produce H
2O
2 in response to a calcium signal. Using a gene-deficient mouse model we also demonstrate that H
2O
2 is produced by the NADPH oxidase Duox1, which is expressed in the mouse urothelium. In contrast, we found no evidence for the expression of lactoperoxidase, an enzyme that has been shown to cooperate with Duox enzymes. We also found that specific activation of TRPV4 calcium channels elicits a calcium signal and stimulates H
2O
2 production in urothelial cells. Furthermore, we detected altered pressure responses in the urinary bladders of Duox1 knockout animals. Our results raise the possibility that mechanosensing in epithelial cells involves calcium-dependent H
2O
2 production similar to that observed in plants.
Ras-related C3 botulinum toxin substrate 2 (RAC2), through interactions with reduced NAD phosphate oxidase component p67phox, activates neutrophil superoxide production, whereas interactions with ...p21-activated kinase are necessary for fMLF-induced actin remodeling. We identified 3 patients with de novo RAC2E62K mutations resulting in severe T- and B-cell lymphopenia, myeloid dysfunction, and recurrent respiratory infections. Neutrophils from RAC2E62K patients exhibited excessive superoxide production, impaired fMLF-directed chemotaxis, and abnormal macropinocytosis. Cell lines transfected with RAC2E62K displayed characteristics of active guanosine triphosphate (GTP)–bound RAC2 including enhanced superoxide production and increased membrane ruffling. Biochemical studies demonstrated that RAC2E62K retains intrinsic GTP hydrolysis; however, GTPase-activating protein failed to accelerate hydrolysis resulting in prolonged active GTP-bound RAC2. Rac2+/E62K mice phenocopy the T- and B-cell lymphopenia, increased neutrophil F-actin, and excessive superoxide production seen in patients. This gain-of-function mutation highlights a specific, nonredundant role for RAC2 in hematopoietic cells that discriminates RAC2 from the related, ubiquitous RAC1.
•E62K is a dominant activating mutation of the small GTPase, RAC2; patients with RAC2E62K have lymphoid and myeloid defects.•Rac2+/E62K mice phenocopy the T- and B-cell lymphopenia, increased neutrophil F-actin, and excessive superoxide production seen in patients.
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Mammalian peroxidases are heme-containing enzymes that serve diverse biological roles, such as host defense and hormone biosynthesis. A mammalian homolog of Drosophila peroxidasin belongs to the ...peroxidase family; however, its function is currently unknown. In this study, we show that peroxidasin is present in the endoplasmic reticulum of human primary pulmonary and dermal fibroblasts, and the expression of this protein is increased during transforming growth factor-β1-induced myofibroblast differentiation. Myofibroblasts secrete peroxidasin into the extracellular space where it becomes organized into a fibril-like network and colocalizes with fibronectin, thus helping to form the extracellular matrix. We also demonstrate that peroxidasin expression is increased in a murine model of kidney fibrosis and that peroxidasin localizes to the peritubular space in fibrotic kidneys. In addition, we show that this novel pathway of extracellular matrix formation is unlikely mediated by the peroxidase activity of the protein. Our data indicate that peroxidasin secretion represents a previously unknown pathway in extracellular matrix formation with a potentially important role in the physiological and pathological fibrogenic response.
Dual oxidases Donkó, Ágnes; Péterfi, Zalán; Sum, Adrienn ...
Philosophical transactions of the Royal Society of London. Series B. Biological sciences,
12/2005, Letnik:
360, Številka:
1464
Journal Article
Recenzirano
Odprti dostop
Reactive oxygen species (ROS) have an important role in various physiological processes including host defence, mitogenesis, hormone biosynthesis, apoptosis and fertilization. Currently, the most ...characterized ROS-producing system operates in phagocytic cells, where ROS generated during phagocytosis act in host defence. Recently, several novel homologues of the phagocytic oxidase have been discovered and this protein family is now designated as the NOX/DUOX family of NADPH oxidases. NOX/DUOX enzymes function in a variety of tissues, including colon, kidney, thyroid gland, testis, salivary glands, airways and lymphoid organs. Importantly, members of the enzyme family are also found in non-mammalian species, including Caenorhabditis elegans and sea urchin. The physiological functions of novel NADPH oxidase enzymes are currently largely unknown. This review focuses on our current knowledge about dual oxidases.
Nox/Duox NADPH oxidases are now considered the primary, regulated sources of reactive oxygen species (ROS). These enzymes are expressed in diverse cells and tissues, and their products are essential ...in several physiological settings. Knockout mouse models are instrumental in identifying the physiological functions of Nox/Duox enzymes as well as in exploring the impact of their pharmacological targeting on disease progression. The currently available data from experiments on knockout animals suggest that the lack of non-phagocytic Nox/Duox enzymes often modifies the course and phenotype in many disease models. Nevertheless, as illustrated by studies on Nox4-deficient animals, the absence of Nox-derived ROS can also lead to aggravated disease manifestation, reinforcing the need for a more balanced view on the role of ROS in health and disease.
Here we describe a 10-year-old girl with combined immunodeficiency presenting as recurring chest infections, lung disease and herpetic skin infections. The patient experienced two hematopoietic stem ...cell transplantations and despite full chimerism, she developed bone marrow aplasia due to adenovirus infection and died at post-transplant day 86. Immunologic investigation revealed low numbers of TRECs/KRECs, a severe reduction of memory B cells, absence of isohemagglutinins, and low IgG levels. Whole exome sequencing (WES) identified a novel heterozygous mutation in RAC2(c.275A > C, p.N92 T). Flow cytometric investigation of neutrophil migration demonstrated an absence of chemotaxis to fMLP. Cell lines transfected with RAC2 N92 T displayed characteristics of active GTP-bound RAC2 including enhanced NADPH oxidase-derived superoxide production both at rest and in response to PMA.
Our findings broaden the clinical picture of RAC2 dysfunction, showing that some individuals can present with a combined immunodeficiency later in childhood rather than a congenital neutrophil disease.
•We report a new case of heterozygous RAC2 deficiency, with the clinical presentation of recurrent respiratory infections, lung disease and susceptibility to varicella and herpetic infections.•Our report expands the phenotypic spectrum of RAC2 deficiency•Dominant activating mutations in RAC2 gene can lead to presentations of combined immunodeficiency where both myeloid and lymphoid lineages are affected
Peroxidases serve diverse biological functions including well-characterized activities in host defence and hormone biosynthesis. More recently, peroxidasin (PXDN) was found to be involved in collagen ...IV cross-linking in the extracellular matrix (ECM). The aim of this study was to characterize the expression and function of peroxidasin-like protein (PXDNL), a previously unknown peroxidase homologue.
We cloned the PXDNL cDNA from the human heart and identified its expression pattern by northern blot, in situ hybridization, and immunohistochemistry. PXDNL is expressed exclusively in the heart and it has evolved to lose its peroxidase activity. The protein is produced by cardiomyocytes and localizes to cell-cell junctions. We also demonstrate that PXDNL can form a complex with PXDN and antagonizes its peroxidase activity. Furthermore, we show an increased expression of PXDNL in the failing myocardium.
PXDNL is a unique component of the heart with a recently evolved inactivation of peroxidase function. The elevation of PXDNL levels in the failing heart may contribute to ECM dysregulation due to its antagonism of PXDN function.
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.
Hydrogen peroxide (H2O2) is an emerging signaling molecule with diverse regulatory functions. Despite its significance, the spatial and temporal organization of H2O2 signals within cells is basically ...unknown. Our limited knowledge about H2O2 signals is largely due to the lack of appropriate techniques for measuring intracellular H2O2. The aim of the current study was to develop novel fluorescent reporter proteins for the measurement of intracellular H2O2.
We developed two novel, fluorescence resonance energy transfer-based redox probes that undergo opposite emission ratio changes upon exposure to H2O2. We have successfully used these sensors to measure H2O2 production by NADPH oxidases (Nox). Moreover, we targeted these probes to specific cellular compartments or incorporated them into oxidase complexes to detect H2O2 at different, well-defined loci.
Studying Nox2- and dual oxidase 1 (Duox1)-expressing cells, we provide the first analysis of how NADPH-oxidase generated H2O2 signals radiate within and between cells.
Our results suggest that H2O2 produced by Noxs can induce redox changes in the intracellular milieu of Nox/Duox-expressing cells while simultaneously transmitting paracrine effects to neighboring cells.
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