Chronic granulomatous disease (CGD) is an inherited disorder of phagocyte function in which defective superoxide production results in deficient microbicidal activity. CGD patients suffer from recurrent, life-threatening infections, and nearly half develop chronic gastrointestinal (GI) complications (colitis, gastric outlet obstruction, or perirectal abscess) and/or autoimmune/rheumatologic disorders (AIDs). To identify genetic modifiers of disease severity, we studied a cohort of 129 CGD patients, in whom seven candidate genes (

Chronic granulomatous disease (CGD) is a hereditary disorder of host defense due to absent or decreased activity of phagocyte NADPH oxidase. The X-linked form of the disease derives from defects in the CYBB gene, which encodes the 91-kD glycoprotein component (termed "gp91-phox") of the oxidase. We have identified the mutations in the CYBB gene responsible for X-linked CGD in 131 consecutive independent kindreds. Screening by SSCP analysis identified mutations in 124 of the kindreds, and sequencing of all exons and intron boundary regions revealed the other seven mutations. We detected 103 different specific mutations; no single mutation appeared in more than seven independent kindreds. The types of mutations included large and small deletions (11%), frameshifts (24%), nonsense mutations (23%), missense mutations (23%), splice-region mutations (17%), and regulatory-region mutations (2%). The distribution of mutations within the CYBB gene exhibited great heterogeneity, with no apparent mutational hot spots. Evaluation of 87 available mothers revealed X-linked carrier status in all but 10. The heterogeneity of mutations and the lack of any predominant genotype indicate that the disease represents many different mutational events, without a founder effect, as is expected for a disorder with a previously lethal phenotype.

Regulated generation of reactive oxygen species (ROS) is primarily accomplished by NADPH oxidases (Nox).Nox1 to Nox4 form a membrane-associated heterodimer with p22 phox , creating the docking site for assembly of the activated oxidase. Signaling specificity is achieved by interaction with a complex network of cytosolic components. Nox4, an oxidase linked to cardiovascular disease, carcinogenesis, and pulmonary fibrosis, deviates from this model by displaying constitutive H 2 O 2 production without requiring known regulators. Extensive Nox4/Nox2 chimera screening was initiated to pinpoint structural motifs essential for ROS generation and Nox subcellular localization. In summary, a matching B loop was crucial for catalytic activity of both Nox enzymes. Substitution of the carboxyl terminus was sufficient for converting Nox4 into a phorbol myristate acetate (PMA)-inducible phenotype, while Nox2-based chimeras never gained constitutive activity. Changing the Nox2 but not the Nox4 amino terminus abolished ROS generation. The unique heterodimerization of a functional Nox4/p22 phox Y121H complex was dependent on the D loop. Nox4, Nox2, and functional Nox chimeras translocated to the plasma membrane. Cell surface localization of Nox4 or PMA-inducible Nox4 did not correlate with O 2 ؊ generation. In contrast, Nox4 released H 2 O 2 and promoted cell migration. Our work provides insights into Nox structure, regulation, and ROS output that will aid inhibitor design.

The integral membrane protein p22phox forms a heterodimeric enzyme complex with NADPH oxidases (Noxs) and is required for their catalytic activity. Nox4, a Nox linked to cardiovascular disease, angiogenesis, and insulin signaling, is unique in its ability to produce hydrogen peroxide constitutively. To date, p22 phox constitutes the only identified regulatory component for Nox4 function. To delineate structural elements in p22 phox essential for formation and localization of the Nox4-p22 phox complex and its enzymatic function, truncation and point mutagenesis was used. Human lung carcinoma cells served as a heterologous expression system, since this cell type is p22 phox -deficient and promotes cell surface expression of the Nox4-p22 phox heterodimer. Expression of p22 phox truncation mutants indicates that the dual tryptophan motif contained in the N-terminal amino acids 6 -11 is essential, whereas the C terminus (amino acids 130 -195) is dispensable for Nox4 activity. Introduction of charged residues in domains predicted to be extracellular by topology modeling was mostly tolerated, whereas the exchange of amino acids in predicted membranespanning domains caused loss of function or showed distinct differences in p22 phox interaction with various Noxs. For example, the substitution of tyrosine 121 with histidine in p22 phox , which abolished Nox2 and Nox3 function in vivo, preserved Nox4 activity when expressed in lung cancer cells. Many of the examined p22 phox mutations inhibiting Nox1 to -3 maturation did not alter Nox4-p22 phox association, further accenting the differences between Noxs. These studies highlight the distinct interaction of the key regulatory p22 phox subunit with Nox4, a feature which could provide the basis for selective inhibitor development.The phagocyte NADPH oxidase consists of two membraneassociated subunits, the catalytic Nox2 (gp91 phox ) and the small subunit p22phox . Upon activation of the oxidase, several regulatory proteins in the cytosol undergo changes, such as incorporation of GTP or phosphorylation, leading to translocation and assembly of a multimeric oxidase complex at the membrane (1-3). This catalytically active complex shuttles electrons across the membrane in order to reduce molecular oxygen to superoxide. Although Nox2 contains the NADPH binding site, the flavin, and the heme groups required for accepting and transferring electrons, electron flow is also dependent on the presence of p22 phox as a focal point for oxidase assembly.

Chronic granulomatous disease (CGD) is a primary immunodeficiency caused by defects in any one of 4 genes encoding phagocyte NADPH oxidase subunits. Unlike other CGD subtypes, in which there is great heterogeneity among mutations, 97% of affected alleles in patients previously reported with A47 0 CGD carry a single mutation, a GT deletion (⌬GT) in exon 2 of the p47-phox gene, NCF-1. This unusually high incidence results from recombination events between NCF-1 and its highly homologous pseudogenes, in which ⌬GT originates. In 50 consecutive patients with A47 0 CGD, 4 were identified who were heterozygous for ⌬GT in NCF-1, and for the first time, 2 were identified whose DNA appeared normal at this position. To avoid co-amplification of pseudogene sequence and to enable the identification of mutations in these patients, allele-specific polymerase chain reaction was used to amplify alleles not containing ⌬GT. In each of the 4 patients who were heterozygous for ⌬GT, an additional novel mutation was identified. These were 2 missense mutations, G125 3 A in exon 2 (predicting Arg42 3 Gln) and G784 3 A in exon 8 (Gly262 3 Ser), and 2 splice junction mutations at the 5 end of intron 1, gt 3 at and gtg 3 gtt. The first of 2 patients who appeared normal at the GT position was a compound heterozygote with the G125 3 A transition on one allele and a deletion of G811 on the other. In the second of these patients, only a single defect was detected, G574 3 A, which predicts Gly192 3 Ser but is likely to result in defective splicing because it represents the final nucleotide of exon 6. (Blood. 2001;97:305-311)

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Duox NADPH oxidases generate hydrogen peroxide at the air-liquid interface of the respiratory tract and at apical membranes of thyroid follicular cells. Inactivating mutations of Duox2 have been linked to congenital hypothyroidism, and epigenetic silencing of Duox is frequently observed in lung cancer. To study Duox regulation by maturation factors in detail, its association with these factors, differential use of subunits and localization was analyzed in a lung cancer cell line and undifferentiated or polarized lung epithelial cells. We show here that Duox proteins form functional heterodimers with their respective DuoxA subunits, in close analogy to the phagocyte NADPH oxidase. Characterization of novel DuoxA1 isoforms and mispaired Duox-DuoxA complexes revealed that heterodimerization is a prerequisite for reactive oxygen species production. Functional Duox1 and Duox2 localize to the leading edge of migrating cells, augmenting motility and wound healing. DuoxA subunits are responsible for targeting functional oxidases to distinct cellular compartments in lung epithelial cells, including Duox2 expression in ciliated cells in an ex vivo differentiated lung epithelium. As these locations probably define signaling specificity of Duox1 versus Duox2, these findings will facilitate monitoring Duox isoform expression in lung disease, a first step for early screening procedures and rational drug development.

Bacillus anthracis lethal toxin (LT) is a key virulence factor of anthrax and contributes significantly to the in vivo pathology. The enzymatically active component is a Zn2+-dependent metalloprotease that cleaves most isoforms of mitogen-activated protein kinase kinases (MKKs). Using ex vivo differentiated human lung epithelium we report that LT destroys lung epithelial barrier function and wound healing responses by immobilizing the actin and microtubule network. Long-term exposure to the toxin generated a unique cellular phenotype characterized by increased actin filament assembly, microtubule stabilization, and changes in junction complexes and focal adhesions. LT-exposed cells displayed randomly oriented, highly dynamic protrusions, polarization defects and impaired cell migration. Reconstitution of MAPK pathways revealed that this LT-induced phenotype was primarily dependent on the coordinated loss of MKK1 and MKK2 signaling. Thus, MKKs control fundamental aspects of cytoskeletal dynamics and cell motility. Even though LT disabled repair mechanisms, agents such as keratinocyte growth factor or dexamethasone improved epithelial barrier integrity by reducing cell death. These results suggest that co-administration of anti-cytotoxic drugs may be of benefit when treating inhalational anthrax.