The Respiratory Burst Oxidase Homolog Protein D (GhRbohD) Positively Regulates the Cotton Resistance to Verticillium dahliae.

Verticillium wilt, mainly caused by a soil-inhabiting fungus Verticillium dahliae, can seriously reduce the yield and quality of cotton. The complex mechanism underlying cotton resistance to Verticillium wilt remains largely unknown. In plants, reactive oxygen species (ROS) mediated by Rbohs is one of the earliest responses of plants to biotic and abiotic stresses. In our previous study, we performed a time-course phospho-proteomic analysis of roots of resistant and susceptible cotton varieties in response to V. dahliae, and found early differentially expressed protein burst oxidase homolog protein D (GhRbohD). However, the role of GhRbohD-mediated ROS in cotton defense against V. dahliae needs further investigation. In this study, we analyzed the function of GhRbohD-mediated resistance of cotton against V. dahliae in vitro and in vivo. Bioinformatics analysis showed that GhRbohD possessed the conservative structural attributes of Rbohs family, 12 members of RbohD out of 57 Rbohs in cotton. The expression of GhRbohD was significantly upregulated after V. dahliae inoculation, peaking at 6 hpi, and the phosphorylation level was also increased. A VIGS test demonstrated that ROS production, NO, H2O2 and Ca2+ contents of GhRbohD-silenced cotton plants were significantly reduced, and lignin synthesis and callose accumulation were damaged, important reasons for the impairment of GhRbohD-silenced cotton's defense against V. dahliae. The expression levels of resistance-related genes were downregulated in GhRbohD-silenced cotton by qRT-PCR, mainly involving the lignin metabolism pathway and the jasmonic acid signaling pathway. However, overexpression of GhRbohD enhanced resistance of transgenic Arabidopsis to V. dahliae challenge. Furthermore, Y2H assays were applied to find that GhPBL9 and GhRPL12C may interact with GhRbohD. These results strongly support that GhRbohD activates ROS production to positively regulate the resistance of plants against V. dahliae.

Molecular insights of NADPH oxidases and its pathological consequences.

Reactive oxygen species (ROS), formed by the partial reduction of oxygen, were for a long time considered to be a byproduct of cellular metabolism. Since, increase in cellular levels of ROS results in oxidative stress leading to damage of nucleic acids, proteins, and lipids resulting in numerous pathological conditions; ROS was considered a bane for aerobic species. Hence, the discovery of NADPH oxidases (NOX), an enzyme family that specifically generates ROS as its prime product came as a surprise to redox biologists. NOX family proteins participate in various cellular functions including cell proliferation and differentiation, regulation of genes and protein expression, apoptosis, and host defence immunological response. Balanced expression and activation of NOX with subsequent production of ROS are critically important to regulate various genes and proteins to maintain homeostasis of the cell. However, dysregulation of NOX activation leading to enhanced ROS levels is associated with various pathophysiologies including diabetes, cardiovascular diseases, neurodegenerative diseases, ageing, atherosclerosis, and cancer. Although our current knowledge on NOX signifies its importance in the normal functioning of various cellular pathways; yet the choice of ROS producing enzymes which can tip the scale from homeostasis toward damage, as mediators of biological functions remain an oddity. Though the role of NOX in maintaining normal cellular functions is now deemed essential, yet its dysregulation leading to catastrophic events cannot be denied. Hence, this review focuses on the involvement of NOX enzymes in various pathological conditions imploring them as possible targets for therapies. SIGNIFICANCE OF THE STUDY: The NOXs are multi-subunit enzymes that generate ROS as a prime product. NOX generated ROS are usually regulated by various molecular factors and play a vital role in different physiological processes. The dysregulation of NOX activity is associated with pathological consequences. Recently, the dynamic proximity of NOX enzymes with different molecular signatures of pathologies has been studied extensively. It is essential to identify the precise role of NOX machinery in its niche during the progression of pathology. Although inhibition of NOX could be a promising approach for therapeutic interventions, it is critical to expand the current understanding of NOX's dynamicity and shed light on their molecular partners and regulators.

NADPH oxidases in bone and cartilage homeostasis and disease: A promising therapeutic target.

Reactive oxygen species (ROS) generated by the NADPH oxidase (Nox) enzymes are important short-range signaling molecules. They have been extensively studied in the physiology and pathophysiology of the cardiovascular system, where they have important roles in vascular inflammation, angiogenesis, hypertension, cardiac injury, stroke, and aging. Increasing evidence demonstrates that ROS and Nox enzymes also affect bone homeostasis and osteoporosis, and more recent studies implicate ROS and Nox enzymes in both inflammatory arthritis and osteoarthritis. Mechanistically, this connection may be through the effects of ROS on signal transduction. ROS affect both transforming growth factor-ß/Smad signaling, interleukin-1ß/nuclear factor-kappa B signaling, and the resulting changes in matrix metalloproteinase expression. The purpose of this review is to describe the role of Nox enzymes in the physiology and pathobiology of bone and joints and to highlight the potential of therapeutically targeting the Nox enzymes.

Comparative analysis of the reactive oxygen species-producing enzymatic activity of Arabidopsis NADPH oxidases.

Reactive oxygen species (ROS) produced by NADPH oxidases, called respiratory burst oxidase homologs (Rbohs), play crucial roles in development as well as biotic and abiotic stress responses in plants. Arabidopsis has 10 Rboh genes, AtRbohA to AtRbohJ. Five AtRbohs (AtRbohC, -D, -F, -H and -J) are synergistically activated by Ca2+ -binding and protein phosphorylation to produce ROS that play various roles in planta, although the activities of the other Rbohs remain unknown. With a heterologous expression system, we found a range of ROS-producing activity among the AtRbohs with differences up to 100 times, indicating that the required amounts of ROS are different in each situation where AtRbohs act. To specify the functions of AtRbohs involved in cell growth, we focused on AtRbohC, -H and -J, which are involved in tip growth of root hairs or pollen tubes. Ectopic expression of the root hair factor AtRbohC/ROOT HAIR DEFECTIVE 2 (RHD2) in pollen tubes restored the atrbohH atrbohJ defects in tip growth of pollen tubes. However, expression of AtRbohH or -J in root hairs did not complement the tip growth defect in the atrbohC/rhd2 mutant. Our data indicate that Rbohs possess different ranges of enzymatic activity, and that some Rbohs have evolved to carry specific functions in cell growth.

Structural complexity and functional diversity of plant NADPH oxidases.

Plant NADPH oxidases also known as respiratory burst oxidase homologs (Rbohs) are a family of membrane-bound enzymes that play diverse roles in the defense response and morphogenetic processes via regulated generation of reactive oxygen species. Rbohs are associated with a variety of functions, although the reason for this is not clear. To evaluate using bioinformatics, the possible mechanisms for the observed functional diversity within the plant kingdom, 127 Rboh protein sequences representing 26 plant species were analyzed. Multiple clusters were identified with gene duplications that were both dicot as well as monocot-specific. The N-terminal sequences were observed to be highly variable. The conserved cysteine (equivalent of Cys890) in C-terminal of AtRbohD suggested that the redox-based modification like S-nitrosylation may regulate the activity of other Rbohs. Three-dimensional models corresponding to the N-terminal domain for Rbohs from Arabidopsis thaliana and Oryza sativa were constructed and molecular dynamics studies were carried out to study the role of Ca2+ in the folding of Rboh proteins. Certain mutations indicated possibly affect the structure and function of the plant NADPH oxidases, thereby providing the rationale for further experimental validation.

Nox NADPH oxidases and the endoplasmic reticulum.

SIGNIFICANCE: Understanding isoform- and context-specific subcellular Nox reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase compartmentalization allows relevant functional inferences. This review addresses the interplay between Nox NADPH oxidases and the endoplasmic reticulum (ER), an increasingly evident player in redox pathophysiology given its role in redox protein folding and stress responses. RECENT ADVANCES: Catalytic/regulatory transmembrane subunits are synthesized in the ER and their processing includes folding, N-glycosylation, heme insertion, p22phox heterodimerization, as shown for phagocyte Nox2. Dual oxidase (Duox) maturation also involves the regulation by ER-resident Duoxa2. The ER is the activation site for some isoforms, typically Nox4, but potentially other isoforms. Such location influences redox/Nox-mediated calcium signaling regulation via ER targets, such as sarcoendoplasmic reticulum calcium ATPase (SERCA). Growing evidence suggests that Noxes are integral signaling elements of the unfolded protein response during ER stress, with Nox4 playing a dual prosurvival/proapoptotic role in this setting, whereas Nox2 enhances proapoptotic signaling. ER chaperones such as protein disulfide isomerase (PDI) closely interact with Noxes. PDI supports growth factor-dependent Nox1 activation and mRNA expression, as well as migration in smooth muscle cells, and PDI overexpression induces acute spontaneous Nox activation. CRITICAL ISSUES: Mechanisms of PDI effects include possible support of complex formation and RhoGTPase activation. In phagocytes, PDI supports phagocytosis, Nox activation, and redox-dependent interactions with p47phox. Together, the results implicate PDI as possible Nox organizer. FUTURE DIRECTIONS: We propose that convergence between Noxes and ER may have evolutive roots given ER-related functional contexts, which paved Nox evolution, namely calcium signaling and pathogen killing. Overall, the interplay between Noxes and the ER may provide relevant insights in Nox-related (patho)physiology.

NADPH oxidases in vascular pathology.

SIGNIFICANCE: Reactive oxygen species (ROS) play a critical role in vascular disease. While there are many possible sources of ROS, nicotinamide adenine dinucleotide phosphate (NADPH) oxidases play a central role. They are a source of "kindling radicals," which affect other enzymes, such as nitric oxide synthase endothelial nitric oxide synthase or xanthine oxidase. This is important, as risk factors for atherosclerosis (hypertension, diabetes, hypercholesterolemia, and smoking) regulate the expression and activity of NADPH oxidases in the vessel wall. RECENT ADVANCES: There are seven isoforms in mammals: Nox1, Nox2, Nox3, Nox4, Nox5, Duox1 and Duox2. Nox1, Nox2, Nox4, and Nox5 are expressed in endothelium, vascular smooth muscle cells, fibroblasts, or perivascular adipocytes. Other homologues have not been found or are expressed at very low levels; their roles have not been established. Nox1/Nox2 promote the development of endothelial dysfunction, hypertension, and inflammation. Nox4 may have a role in protecting the vasculature during stress; however, when its activity is increased, it may be detrimental. Calcium-dependent Nox5 has been implicated in oxidative damage in human atherosclerosis. CRITICAL ISSUES: NADPH oxidase-derived ROS play a role in vascular pathology as well as in the maintenance of normal physiological vascular function. We also discuss recently elucidated mechanisms such as the role of NADPH oxidases in vascular protection, vascular inflammation, pulmonary hypertension, tumor angiogenesis, and central nervous system regulation of vascular function and hypertension. FUTURE DIRECTIONS: Understanding the role of individual oxidases and interactions between homologues in vascular disease is critical for efficient pharmacological regulation of vascular NADPH oxidases in both the laboratory and clinical practice.

Evolutionary dynamics of the human NADPH oxidase genes CYBB, CYBA, NCF2, and NCF4: functional implications.

The phagocyte NADPH oxidase catalyzes the reduction of O2 to reactive oxygen species with microbicidal activity. It is composed of two membrane-spanning subunits, gp91-phox and p22-phox (encoded by CYBB and CYBA, respectively), and three cytoplasmic subunits, p40-phox, p47-phox, and p67-phox (encoded by NCF4, NCF1, and NCF2, respectively). Mutations in any of these genes can result in chronic granulomatous disease, a primary immunodeficiency characterized by recurrent infections. Using evolutionary mapping, we determined that episodes of adaptive natural selection have shaped the extracellular portion of gp91-phox during the evolution of mammals, which suggests that this region may have a function in host-pathogen interactions. On the basis of a resequencing analysis of approximately 35 kb of CYBB, CYBA, NCF2, and NCF4 in 102 ethnically diverse individuals (24 of African ancestry, 31 of European ancestry, 24 of Asian/Oceanians, and 23 US Hispanics), we show that the pattern of CYBA diversity is compatible with balancing natural selection, perhaps mediated by catalase-positive pathogens. NCF2 in Asian populations shows a pattern of diversity characterized by a differentiated haplotype structure. Our study provides insight into the role of pathogen-driven natural selection in an innate immune pathway and sheds light on the role of CYBA in endothelial, nonphagocytic NADPH oxidases, which are relevant in the pathogenesis of cardiovascular and other complex diseases.

NADPH Oxidases NOXs and DUOXs as putative targets for cancer therapy.

Reactive oxygen species (ROS) form a class of molecules with both positive and negative impacts on cellular health. Negatively, ROS may react with cellular constituents including proteins, lipids, and DNA to generate an array of oxidative lesions. These lesions may compromise genome stability which is critical for long-term cellular homeostasis and healthy progeny. Paradoxically, ROS also function as strong signalling molecules that mediate various growth-related responses, so their presence is also essential for cellular metabolism. While ROS are generated in an unregulated manner by physical stresses such as exposure to ionizing radiation and biochemical malfunctions such as mitochondrial leakage, cells also contain the NADPH oxidases NOXs and DUOXs, which specifically generate ROS in a wide variety of tissues. While the NOXs/DUOXs may be involved in maintaining optimal cellular redox levels, there is also accumulating evidence that NADPH oxidases-derived ROS may elevate the risk for genomic instability and cancer. Cancer cells may produce high levels of ROS, and in some cases, the source of these ROS has been linked to NOX/DUOX deregulation as reported for prostate cancer (NOX1 and NOX5), melanoma and glioblastoma (NOX4) among others. In addition, recent studies reveal that targeting NADPH oxidases with NOXs inhibitors may impair tumor growth in vivo; indicating that these proteins may be useful targets in future clinical strategies to fight cancer. This review provides an overview of the current knowledge concerning these enzymes, their roles in cancer, and their potential as targets in future cancer therapies.

The NADPH oxidase family and its inhibitors.

The classical nicotinamide adenine dinucleotide phosphate (NADPH) oxidase was originally detected in neutrophils as a multicomponent enzyme that catalyzes the generation of superoxide from oxygen and the reduced form of NADPH. This enzyme is composed of two membrane-bound subunits (p22phox and gp91phox), three cytosolic subunits (p67phox, p47phox, and p40phox) and a small G-protein Rac (Rac1 and Rac2). Recently, it has been demonstrated that there are several isoforms of nonphagocytic NADPH oxidase. Endothelial cells, vascular smooth muscle cells or adventitial fibroblasts possess multiple isoforms of this enzyme. The new homologs, along with gp91phox are now designated the Nox family of NADPH oxidases and are key sources of reactive oxygen species in the vasculature. Reactive oxygen species play a significant role in regulating endothelial function and vascular tone. However, besides the participation in the processes of physiological cell, these enzymes can also be the perpetrator of oxidative stress that causes endothelial dysfunction. This review summarizes the current state of knowledge of the structure and functions of NADPH oxidase and NADPH oxidase inhibitors in the treatment of disorders with endothelial damage.

Yno1p/Aim14p, a NADPH-oxidase ortholog, controls extramitochondrial reactive oxygen species generation, apoptosis, and actin cable formation in yeast.

The large protein superfamily of NADPH oxidases (NOX enzymes) is found in members of all eukaryotic kingdoms: animals, plants, fungi, and protists. The physiological functions of these NOX enzymes range from defense to specialized oxidative biosynthesis and to signaling. In filamentous fungi, NOX enzymes are involved in signaling cell differentiation, in particular in the formation of fruiting bodies. On the basis of bioinformatics analysis, until now it was believed that the genomes of unicellular fungi like Saccharomyces cerevisiae and Schizosaccharomyces pombe do not harbor genes coding for NOX enzymes. Nevertheless, the genome of S. cerevisiae contains nine ORFs showing sequence similarity to the catalytic subunits of mammalian NOX enzymes, only some of which have been functionally assigned as ferric reductases involved in iron ion transport. Here we show that one of the nine ORFs (YGL160W, AIM14) encodes a genuine NADPH oxidase, which is located in the endoplasmic reticulum (ER) and produces superoxide in a NADPH-dependent fashion. We renamed this ORF YNO1 (yeast NADPH oxidase 1). Overexpression of YNO1 causes YCA1-dependent apoptosis, whereas deletion of the gene makes cells less sensitive to apoptotic stimuli. Several independent lines of evidence point to regulation of the actin cytoskeleton by reactive oxygen species (ROS) produced by Yno1p.

Dual oxidase in the intestinal epithelium of zebrafish larvae has anti-bacterial properties.

Reactive oxygen species (ROS) function in a range of physiological processes such as growth, metabolism and signaling, and also have a pathological role. Recent research highlighted the requirement for ROS generated by dual oxidase (DUOX) in host-defence responses in innate immunity and inflammatory disorders such as inflammatory bowel disease (IBD), but in vivo evidence to support this has, to date, been lacking. In order to investigate the involvement of Duox in gut immunity, we characterized the zebrafish ortholog of the human DUOX genes. Zebrafish duox is highly expressed in intestinal epithelial cells. Knockdown of Duox impaired larval capacity to control enteric Salmonella infection.

The NADPH oxidase Cpnox1 is required for full pathogenicity of the ergot fungus Claviceps purpurea.

The role of reactive oxygen species (ROS) in interactions between phytopathogenic fungi and their hosts is well established. An oxidative burst mainly caused by superoxide formation by membrane-associated NADPH oxidases is an essential element of plant defence reactions. Apart from primary effects, ROS play a major role as a second messenger in host response. Recently, NADPH oxidase (nox)-encoding genes have been identified in filamentous fungi. Functional analyses have shown that these fungal enzymes are involved in sexual differentiation, and there is growing evidence that they also affect developmental programmes involved in fungus-plant interactions. Here we show that in the biotrophic plant pathogen Claviceps purpurea deletion of the cpnox1 gene, probably encoding an NADPH oxidase, has impact on germination of conidia and pathogenicity: Deltacpnox1 mutants can penetrate the host epidermis, but they are impaired in colonization of the plant ovarian tissue. In the few cases where macroscopic signs of infection (honeydew) appear, they are extremely delayed and fully developed sclerotia have never been observed. C. purpurea Nox1 is important for the interaction with its host, probably by directly affecting pathogenic differentiation of the fungus.

NADPH oxidases are involved in differentiation and pathogenicity in Botrytis cinerea.

Nicotinamide adenine dinucleotide (NADPH) oxidases have been shown to be involved in various differentiation processes in fungi. We investigated the role of two NADPH oxidases in the necrotrophic phytopathogenic fungus, Botrytis cinerea. The genes bcnoxA and bcnoxB were cloned and characterized; their deduced amino acid sequences show high homology to fungal NADPH oxidases. Analyses of single and double knock-out mutants of both NADPH oxidase genes showed that both bcnoxA and bcnoxB are involved in formation of sclerotia. Both genes have a great impact on pathogenicity: whereas bcnoxB mutants showed a retarded formation of primary lesions, probably due to an impaired formation of penetration structures, bcnoxA mutants were able to penetrate host tissue in the same way as the wild type but were much slower in colonizing the host tissue. Double mutants showed an additive effect: they were aberrant in penetration and colonization of plant tissue and, therefore, almost nonpathogenic. To study the structure of the fungal Nox complex in more detail, bcnoxR (encoding a homolog of the mammalian p67(phox), a regulatory subunit of the Nox complex) was functionally characterized. The phenotype of DeltabcnoxR mutants is identical to that of DeltabcnoxAB double mutants, providing evidence that BcnoxR is involved in activation of both Bcnox enzymes.

Tissue distribution and putative physiological function of NOX family NADPH oxidases.

The NOX family of ROS-generating NADPH oxidases consists of 7 members: NOX1 to NOX5, DUOX1 and 2. NOX1 is predominantly found in the colon, where it possibly plays a role in the host defense. NOX2 is the phagocyte NADPH oxidase, a clearly established host defense enzyme. NOX3 is almost exclusively expressed in the inner ear, where it is involved in otoconia morphogenesis, but based on its localization might also play a role in the auditory system. NOX4, widely expressed in kidney, vascular cells, osteoclasts etc.; it might be a constitutively active enzyme, regulated on the level of gene expression but its precise physiological function remains unknown. NOX5, a Ca2+ activated enzyme is predominantly expressed in lymphoid tissues and testis, where it might be involved in signaling processes. DUOX1 is expressed in the thyroid and in respiratory epithelia, and DUOX2 in the thyroid and in gastrointestinal glandular epithelia. Both DUOX enzymes are involved in thyroid hormone synthesis, but possibly also in epithelial host defense.

Reactive oxygen species generated by microbial NADPH oxidase NoxA regulate sexual development in Aspergillus nidulans.

NADPH oxidases (Nox) have been characterized as higher eukaryotic enzymes used deliberately to produce reactive oxygen species (ROS). The recent discovery of new functional members of the Nox family in plants and animals has led to the recognition of the increasing importance of ROS as signals involved in regulation of diverse cellular processes such as defence, growth and signalling. Here, we address the role of NADPH oxidase-generated ROS in the biology of the filamentous fungus Aspergillus nidulans. We characterize the noxA gene and show that it encodes a member of a novel NADPH oxidase subfamily ubiquitous in lower eukaryotes. Deletion of noxA specifically blocks differentiation of sexual fruit bodies (cleistothecia), without affecting hyphal growth or asexual development. Accordingly, the noxA gene is induced during sexual development, peaking at the time of cleistothecia differentiation and in parallel with the hülle cell-associated catalase peroxidase gene cpeA. This expression pattern is not dependent on transcription factors SteA and StuA, which are essential for cleistothecia formation. In contrast, noxA-dependent premature sexual development correlates with noxA derepression in DeltasakA null mutants, connecting stress MAPK signalling to the regulated production of ROS. Using a nitroblue tetrazolium (NBT) assay to detect superoxide, we found that hülle cells and cleistothecia initials produce superoxide in a process inhibited by NADPH oxidase inhibitor DPI and markedly reduced in DeltanoxA mutants. Furthermore, using H2DCFDA, we detected that H2O2 and possibly other ROS are generated in a NoxA-dependent fashion, mainly in the external walls from cleistothecia initials. The essential role of NoxA-generated ROS in A. nidulans sexual differentiation and the presence of one or two noxA homologues in all analysed filamentous fungi suggest that NADPH oxidase-generated ROS play important roles in fungal physiology and differentiation.