High molecular weight homologues of gp91phox, the superoxide-generating subunit of phagocyte nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase, have been identified in human (h) and Caenorhabditis elegans (Ce), and are termed Duox for “dual oxidase” because they have both a peroxidase homology domain and a gp91phox domain. A topology model predicts that the enzyme will utilize cytosolic NADPH to generate reactive oxygen, but the function of the ecto peroxidase domain was unknown. Ce-Duox1 is expressed in hypodermal cells underlying the cuticle of larval animals. To investigate function, RNA interference (RNAi) was carried out in C. elegans. RNAi animals showed complex phenotypes similar to those described previously in mutations in collagen biosynthesis that are known to affect the cuticle, an extracellular matrix. Electron micrographs showed gross abnormalities in the cuticle of RNAi animals. In cuticle, collagen and other proteins are cross-linked via di- and trityrosine linkages, and these linkages were absent in RNAi animals. The expressed peroxidase domains of both Ce-Duox1 and h-Duox showed peroxidase activity and catalyzed cross-linking of free tyrosine ethyl ester. Thus, Ce-Duox catalyzes the cross-linking of tyrosine residues involved in the stabilization of cuticular extracellular matrix.

Redox mechanisms function in control of gene expression, cell proliferation, and apoptosis, but the circuitry for redox signaling remains unclear. Cysteine and methionine are the only amino acids in proteins that undergo reversible oxidation/reduction under biologic conditions and, as such, provide a means for control of protein activity, protein-protein interaction, protein trafficking, and protein-DNA interaction. Hydrogen peroxide and other reactive oxygen species (ROS) provide a mechanism to oxidize signaling proteins. However, oxidation of sulfur-containing side chains of cysteine and methionine by ROS can result in oxidation states of sulfur (e.g., sulfinate, sulfonate, sulfone) that are not reducible under biologic conditions. Thus, mechanisms for oxidation that protect against over-oxidation of these susceptible residues and prevent irreversible loss of activity would be advantageous. The present study shows that the steady-state redox potential of the cysteine/cystine couple (Eh = -145 mV) in cells is sufficiently oxidized (>90 mV) relative to the GSH/GSSG (-250 mV) and thioredoxin (Trx1, -280 mV) redox couples for the cysteine/cystine couple to function as an oxidant in redox switching. Consequently, the cysteine/cystine couple provides a means to oxidize proteins without direct involvement of more potent oxidants. A circuitry model incorporating cysteine as a redox node, along with Trx1 and GSH, reveals how selective interactions between the different thiol/disulfide couples and reactive protein thiols could differentially regulate metabolic functions. Moreover, inclusion of cysteine/cystine as a signaling node distinct from GSH and Trx1 significantly expands the redox range over which protein thiol/disulfide couples may operate to control physiologically relevant processes.

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Defensins are a newly recognized class of small, cationic polypeptides that have in vitro microbicidal activity toward certain bacteria, fungi, and viruses. Human neutrophil granules were separated into 13 density fractions by using a high-resolution Percoll gradient centrifugation procedure, and the distribution of the three defensin polypeptides in these fractions was determined. Levels of defensins and several granule marker proteins were estimated in each fraction from relative staining intensities of bands following acid-urea and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of total acid-extractable proteins. These results were confirmed by enzyme immunoassay measurements of defensins and quantitative determinations of the typical azurophil granule components, myeloperoxidase, beta- glucuronidase, lysozyme, and elastase. The five higher density granule fractions (H1 through H5) contained fourfold higher relative amounts of defensins as compared with the eight lower density fractions (L1 through L8), accounting for approximately 50% of the total protein. In particular, fraction H5 was especially enriched in defensins but was relatively deficient in myeloperoxidase, beta-glucuronidase, lysozyme, and elastase. Ultrastructural morphology showed that fraction H5 contained the largest granules. Seventy percent of these granules exhibited electron-dense rims and electron-lucent central regions when stained with methanolic uranyl acetate-lead citrate, and 70% showed this same characteristic rim-staining pattern after limited reaction (30 minutes) for peroxidase with diaminobenzidine. These distinctively large, rim-stained granules were identified in intact, mature peripheral blood neutrophils as well as in human bone marrow promyelocytes, indicating that their synthesis occurs during early myeloid development. This unusual granule type may play a specialized role in the microbicidal functions of the neutrophil, distinct from that of typical azurophil granules.

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