DUOX1 in mammalian disease pathophysiology.

Dual oxidase 1 (DUOX1) is a member of the protein family of nicotinamide adenine dinucleotide phosphate (NADPH) oxidases. DUOX1 has several normal physiological, immunological, and biochemical functions in different parts of the body. Dysregulated oxidative metabolism interferes with various disease pathologies and numerous therapeutic options are based on targeting cellular redox pathways. DUOX1 forms an important enzymatic source of biological oxidants, and DUOX1 expression is frequently dysregulated in various diseases. While this review shortly addresses the biochemical and cellular properties and proposed physiological roles of DUOX1, its main purpose is to summarize the current knowledge with respect to the potential role of DUOX1 enzyme in disease pathology, especially in mammalian organisms. Although DUOX1 is normally prominently expressed in epithelial lineages, it is frequently silenced in epithelial-derived cancers by epigenetic mechanisms. While an abundance of information is available on DUOX1 transcription in different diseases, an increasing number of mechanistic studies indicate a causative relationship between DUOX1 function and disease pathophysiology. Additionally, specific functions of the DUOX1 maturation factor, DUOXA1, will also be addressed. Lastly, urgent and outstanding questions on the field of DUOX1 will be discussed that could provide valuable new diagnostic tools and novel therapeutic options.

Structures of human dual oxidase 1 complex in low-calcium and high-calcium states.

Dual oxidases (DUOXs) produce hydrogen peroxide by transferring electrons from intracellular NADPH to extracellular oxygen. They are involved in many crucial biological processes and human diseases, especially in thyroid diseases. DUOXs are protein complexes co-assembled from the catalytic DUOX subunits and the auxiliary DUOXA subunits and their activities are regulated by intracellular calcium concentrations. Here, we report the cryo-EM structures of human DUOX1-DUOXA1 complex in both high-calcium and low-calcium states. These structures reveal the DUOX1 complex is a symmetric 2:2 hetero-tetramer stabilized by extensive inter-subunit interactions. Substrate NADPH and cofactor FAD are sandwiched between transmembrane domain and the cytosolic dehydrogenase domain of DUOX. In the presence of calcium ions, intracellular EF-hand modules might enhance the catalytic activity of DUOX by stabilizing the dehydrogenase domain in a conformation that allows electron transfer.

PGE2 upregulates gene expression of dual oxidase in a lepidopteran insect midgut via cAMP signalling pathway.

In insect midgut, prostaglandins (PGs) play a crucial role in defending bacterial and malarial pathogens. However, little is known about the PG signalling pathway in the midgut. A dual oxidase (Se-Duox) with presumed function of catalysing reactive oxygen species (ROS) production in the midgut was identified in beet armyworm, Spodoptera exigua. Se-Duox was expressed in all developmental stages, exhibiting relatively high expression levels in the midgut of late larval instars. Se-Duox expression was upregulated upon bacterial challenge. RNA interference (RNAi) of Se-Duox expression significantly suppressed ROS levels in the midgut lumen. The suppression of ROS levels increased insecticidal activity of Serratia marcescens after oral infection. Interestingly, treatment with a PLA2 inhibitor prevented the induction of Se-Duox expression in response to bacterial challenge. On the other hand, addition of its catalytic product rescued the induction of Se-Duox expression. Especially, PG synthesis inhibitor significantly suppressed Se-Duox expression, while the addition of PGE2 or PGD2 rescued the inhibition. Subsequent PG signals involved cAMP and downstream components because specific inhibitors of cAMP signal components such as adenylate cyclase (AC) and protein kinase A (PKA) significantly inhibited Se-Duox expression. Indeed, addition of a cAMP analogue stimulated Se-Duox expression in the midgut. Furthermore, individual RNAi specific to PGE2 receptor (a trimeric G-protein subunit), AC, PKA or cAMP-responsive element-binding protein resulted in suppression of Se-Duox expression. These results suggest that PGs can activate midgut immunity via cAMP signalling pathway by inducing Se-Duox expression along with increased ROS levels.

Structures of mouse DUOX1-DUOXA1 provide mechanistic insights into enzyme activation and regulation.

DUOX1, an NADPH oxidase family member, catalyzes the production of hydrogen peroxide. DUOX1 is expressed in various tissues, including the thyroid and respiratory tract, and plays a crucial role in processes such as thyroid hormone biosynthesis and innate host defense. DUOX1 co-assembles with its maturation factor DUOXA1 to form an active enzyme complex. However, the molecular mechanisms for activation and regulation of DUOX1 remain mostly unclear. Here, I present cryo-EM structures of the mammalian DUOX1-DUOXA1 complex, in the absence and presence of substrate NADPH, as well as DUOX1-DUOXA1 in an unexpected dimer-of-dimers configuration. These structures reveal atomic details of the DUOX1-DUOXA1 interaction, a lipid-mediated NADPH-binding pocket and the electron transfer path. Furthermore, biochemical and structural analyses indicate that the dimer-of-dimers configuration represents an inactive state of DUOX1-DUOXA1, suggesting an oligomerization-dependent regulatory mechanism. Together, my work provides structural bases for DUOX1-DUOXA1 activation and regulation.

Purification and Characterization of DUOX Peroxidase Homology Domains (PHDs).

The dual oxidase (DUOX) enzymes (DUOX1 and DUOX2) are unique hydrogen peroxide (H2O2)-producing members of the NADPH oxidase (NOX) family, structurally distinguished from their related NOX isoforms by the presence of an additional N-terminal extracellular domain. This region has significant sequence and predicted structural homology to mammalian peroxidases, including myeloperoxidase (MPO) and lactoperoxidase (LPO), therefore justifying the nomenclature of the peroxidase homology domain (PHD). Obtaining detailed structural information and defining a function for this appended region are both critical for elucidation of the uncharacterized mechanism of H2O2 production by DUOX proteins. Purification strategies focused on isolated sections of each DUOX enzyme are a logical means to further characterization, particularly as isolation of the complete membrane-bound enzyme in significant quantities remains unachievable. In this chapter, a reproducible method for production of the homology domain applicable to both human DUOX isoforms is described. The approach utilizes a baculovirus expression vector in insect cell culture to produce secreted recombinant PHD; an appended C-terminal His6 affinity tag was found to be crucial for structural stability. Finally, initial characterization of the activity of the purified PHDs is also described.

Functional Characterization of DUOX Enzymes in Reconstituted Cell Models.

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.

Functional Annotation and Analysis of Dual Oxidase 1 (DUOX1): a Potential Anti-pyocyanin Immune Component.

Dual Oxidase 1 (DUOX1) is a prominent immune system component primarily expressed in esophagus, lungs, skin, and urinary bladder including others. DUOX1 is involved in lactoperoxidase-mediated innate immunity at mucosal surfaces by generation of antimicrobial hypothiocyanite at the apical surface of epithelial lining. Upon detection of bacterial pathogens mainly Pseudomonas aeruginosa, DUOX1 is activated in bronchial epithelial cells. Both the host and pathogen enter a redox dual with DUOX1 and hypothiocyanite from host and Pyocyanin (PCN) as a redox active virulence factor from P. aeruginosa. The synergy of the both enzymes permanently oxidizes PCN and thus holds the potential to prevent PCN-induced virulence, which otherwise paves the way for establishment of persistent chronic infection. In this study, we structurally and functionally annotated the DUOX1, predicted its 3d structure, physio-chemical properties, post-translational modifications, and genetic polymorphism analysis with subsequent disease-associated single-nucleotide variations and their impact on DUOX1 functionality by employing in silico approaches. DUOX1 holds greater homology with gorilla and chimpanzee than other primates. The localization signal peptide was present at the beginning of the peptide with cleavage site at 22 aa position. Three distinct functional domains were observed based on homology: An_peroxidase, FRQ1, and oxido-reductase domains. Polymorphism analysis revealed > 60 SNPs associated with different cancers with probable damaging effects. No cancer-associated methylated island was observed for DUOX1. Three-dimensional structure was developed via homology modeling strategy. The proper annotation will help in characterization of DUOX1 and enhance our knowledge of its functionality and biological roles.

Intranasal delivery of Duox2 DNA using cationic polymer can prevent acute influenza A viral infection in vivo lung.

We studied the contribution of Duox2 in mucosal host defense against influenza A virus (IAV) infection in in vivo lung. We found that Duox2 was required for the induction of type I and III interferon (IFN)s and transient Duox2 overexpression using cationic polymer polyethyleneimine (PEI) leads to suppression of IAV infection in in vivo lung. Twenty mice (C57BL/6J) were anesthetized and challenged by intranasal administration of 213 pfu/30 µl of IAV (WS/33/H1N1), and IAV-infected mice were euthanized at 1, 3, 5, 7, 10, 14 days post infection (dpi). Duox2 small hairpin RNA (shRNA) and pCMV-Duox2 formulated with PEI were inoculated to mice to assess the regulatory mechanism between Duox2 and IFN secretion. Following intranasal IAV inoculation, viral infection was significantly aggravated from 3 dpi in in vivo lung and viral titer was highest at 7 dpi. Consistent with this, Duox2 messenger RNA (mRNA) and protein expressions were significantly induced from 3 dpi in the lung tissue of IAV-infected mice. Viral titer was much higher in IAV-infected mice that were inoculated with Duox2 shRNA accompanied with lower survival rate and extensive lung pathologies. Interestingly, severe lung pathologies in IAV-infected mice were not observed and viral titer was significantly reduced in mice with pulmonary administration of pCMV-Duox2 formulated with PEI before IAV inoculation. Both mRNA and secreted protein levels of IFN-ß and IFN-λ2/3 were highly elevated in IAV-infected mice with pCMV-Duox2 formulated with PEI. Duox2 is necessary for the regulation of IFN secretion in in vivo lung, and pulmonary administration of Duox2 DNA using cationic polymer triggers the induction of type I and III IFNs resulting in more complete suppression of IAV infection.

DUOX2 Mutations Are Associated With Congenital Hypothyroidism With Ectopic Thyroid Gland.

Context: Thyroid dysgenesis (TD) is the leading cause of congenital hypothyroidism (CH). The etiology of TD remains unknown in ∼90% of cases, the most common form being thyroid ectopia (TE) (48% to 61%). Objective: To search for candidate genes in hypothyroid children with TE. Design, Setting, and Participants: We followed a cohort of 268 children with TD and performed whole-exome sequencing (WES) in three children with CH with TE (CHTE) and compared them with 18 thyroid-healthy controls. We then screened an additional 41 children with CHTE by Sanger sequencing and correlated the WES and Sanger molecular findings with in vitro functional analysis. Main Outcome Measures: Genotyping, mutation prediction analysis, and in vitro functional analysis. Results: We identified seven variants in the DUOX2 gene, namely G201E, L264CfsX57, P609S, M650T, E810X, M822V, and E1017G, and eight known variations. All children carrying DUOX2 variations had high thyroid-stimulating hormone levels at neonatal diagnosis. All mutations were localized in the N-terminal segment, and three of them led to effects on cell surface targeting and reactive oxygen species generation. The DUOX2 mutants also altered the interaction with the maturation factor DUOXA2 and the formation of a stable DUOX2/DUOXA2 complex at the cell surface, thereby impairing functional enzymatic activity. We observed no mutations in the classic genes related to TD or in the DUOX1 gene. Conclusion: Our findings suggest that, in addition to thyroid hormonogenesis, the DUOX2 N-terminal domain may play a role in thyroid development.