The extracellular A-loop of dual oxidases affects the specificity of reactive oxygen species release

NADPH oxidase (Nox) family proteins produce superoxide (O2.-) directly by transferring an electron to molecular oxygen. Dual oxidases (Duoxes) also produce an O2.- intermediate, although the final species secreted by mature Duoxes is H2O2, suggesting that intramolecular O2.- dismutation or other mechanisms contribute to H2O2 release. We explored the structural determinants affecting reactive oxygen species formation by Duox enzymes. Duox2 showed O2.- leakage when mismatched with Duox activator 1 (DuoxA1). Duox2 released O2.- even in correctly matched combinations, including Duox2 + DuoxA2 and Duox2 + N-terminally tagged DuoxA2 regardless of the type or number of tags. Conversely, Duox1 did not release O2.- in any combination. Chimeric Duox2 possessing the A-loop of Duox1 showed no O2.- leakage; chimeric Duox1 possessing the A-loop of Duox2 released O2.-. Moreover, Duox2 proteins possessing the A-loops of Nox1 or Nox5 co-expressed with DuoxA2 showed enhanced O2.- release, and Duox1 proteins possessing the A-loops of Nox1 or Nox5 co-expressed with DuoxA1 acquired O2.- leakage. Although we identified Duox1 A-loop residues (His1071, His1072, and Gly1074) important for reducing O2.- release, mutations of these residues to those of Duox2 failed to convert Duox1 to an O2.- releasing enzyme. Using immunoprecipitation and endoglycosidase H sensitivity assays, we found that the A-loop of Duoxes binds to DuoxA N termini, creating more stable, mature Duox-DuoxA complexes. In conclusion, the A-loops of both Duoxes support H2O2 production through interaction with corresponding activators, but complex formation between the Duox1 A-loop and DuoxA1 results in tighter control of H2O2 release by the enzyme complex. © 2015, American Society for Biochemistry and Molecular Biology Inc

DUOX Defects and Their Roles in Congenital Hypothyroidism.

Extracellular hydrogen peroxide is required for thyroperoxidase-mediated thyroid hormone synthesis in the follicular lumen of the thyroid gland. Among the NADPH oxidases, dual oxidases, DUOX1 and DUOX2, constitute a distinct subfamily initially identified as thyroid oxidases, based on their level of expression in the thyroid. Despite their high sequence similarity, the two isoforms present distinct regulations, tissue expression, and catalytic functions. Inactivating mutations in many of the genes involved in thyroid hormone synthesis cause thyroid dyshormonogenesis associated with iodide organification defect. This chapter provides an overview of the genetic alterations in DUOX2 and its maturation factor, DUOXA2, causing inherited severe hypothyroidism that clearly demonstrate the physiological implication of this oxidase in thyroid hormonogenesis. Mutations in the DUOX2 gene have been described in permanent but also in transient forms of congenital hypothyroidism. Moreover, accumulating evidence demonstrates that the high phenotypic variability associated with altered DUOX2 function is not directly related to the number of inactivated DUOX2 alleles, suggesting the existence of other pathophysiological factors. The presence of two DUOX isoforms and their corresponding maturation factors in the same organ could certainly constitute an efficient redundant mechanism to maintain sufficient H2O2 supply for iodide organification. Many of the reported DUOX2 missense variants have not been functionally characterized, their clinical impact in the observed phenotype remaining unresolved, especially in mild transient congenital hypothyroidism. DUOX2 function should be carefully evaluated using an in vitro assay wherein (1) DUOXA2 is co-expressed, (2) H2O2 production is activated, (3) and DUOX2 membrane expression is precisely analyzed.info:eu-repo/semantics/publishe

Reactive oxygen species, vascular disease, and hypertension

Reactive oxygen species (ROS) influence many physiological processes including host defense, hormone biosynthesis, fertilization, and cellular signaling. Increased ROS bioavailability and altered redox signaling (oxidative stress) have been implicated in chronic diseases including atherosclerosis and hypertension. Although oxidative injury may not be the sole etiology of hypertension, it amplifies blood pressure elevation in the presence of other pro-hypertensive factors, such as salt loading, activation of the renin-angiotensin system, and sympathetic hyperactivity. Oxidative stress is a multisystem phenomenon in hypertension and involves the heart, kidneys, nervous system, and vessels. A major source for cardiovascular, renal, and neural ROS is a family of non-phagocytic NADPH oxidases, including the prototypic Nox2 homologue-based NADPH oxidase, as well as other NADPH oxidases, such as Nox1 and Nox4. Other possible sources include mitochondrial electron transport enzymes, xanthine oxidase, cyclooxygenase, lipoxygenase, and uncoupled nitric oxide synthase (NOS). Cross talk between Noxes and mitochondrial oxidases is increasingly implicated in cellular ROS production. Convincing findings from experimental and animal studies support a causative role for oxidative stress in the pathogenesis of hypertension. However, there is still no solid evidence that oxidative stress is fundamentally involved in the pathogenesis of human hypertension. Reasons for this are complex and relate to heterogeneity of populations studied, inappropriate or insensitive methodologies to evaluate oxidative state clinically, and suboptimal antioxidant therapies used. Nevertheless, what is becoming increasingly evident is that oxidative stress is important in the molecular mechanisms associated with cardiovascular and renal injury in hypertension and that hypertension itself can contribute to oxidative stress. This chapter provides a comprehensive review of the role of ROS in the (patho)physiology of vascular injury and discusses the importance of Noxes in vascular oxidative stress. Implications in experimental and human hypertension are highlighted