Molecular and Cellular Biology

NADPH Oxidases

The thyroid and hydrogen peroxide

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The main function of the thyroid is the uptake and concentration of iodide from the bloodstream, through the Na+/I- symporteur (NIS) for thyroid hormone biosynthesis. Thyroid hormones, tri- and tetra-iodothyronine (T3 and T4), results from thyroperoxidase (TPO)-catalyzed iodination of thyroglobulin (TG) tyrosine residues and coupling of iodotyrosines. The thyroid hormones act on multiple organs in the body and influence critical physiological processes. They increase the metabolic rate and affect protein synthesis influencing the overall thermoregulation of the human body. They are required for the normal growth and neuronal maturation preventing cretinism. 

Hydrogen peroxide is essential for this hormonogenesis. In the nineties, Bernard Corvilain has demonstrated that, under physiological iodide supply, hormonogenesis is rate-limited by the availability of H2O2. An H2O2-generating system was predicted to exist in the thyroid gland, and the molecular nature of its components has been revealed by Xavier De Deken in 2000 during his PhD. Based on functional homologies with the H2O2-generating system of the leukocyte, two closely related proteins have been identified, the Dual Oxidases (DUOX) 1 and 2. These enzymes are localized with the TPO at the apical pole of the thyrocytes facing the follicular lumen and generate H2O2 outside the cells.

 

 

Biochemical characterization of the NADPH oxidases DUOX1/2: Tight regulation of H2O2 generation

 

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Hydrogen peroxide is required for the thyroid hormone synthesis, but reactive oxygen species (ROS) accumulation could cause irreversible cellular damages. The functional activity of these H2O2 generators DUOX is tightly regulated. The main activator is calcium via direct binding to the proteins. However, recent studies performed in the laboratory have clearly revealed additional mechanisms governing their intrinsic activity via specific protein kinase-mediated phosphorylation which are different between DUOX1 and DUOX2. 

 

 

 

  

 

 

D2DA2-2
 
 

The maturation process of the DUOX isoenzymes constitutes a critical step to acquire their active conformation. In 2006, the DUOX maturation factors have been identified by Dr. H. Grasberger (Chicago University). DUOXA1 and DUOXA2 are N-glycosylated proteins permitting the endoplasmic reticulum exit of properly folded DUOX enzymes. Our research group tries to elucidate the mechanisms governing the DUOX processing allowing the correct addressing of the protein at the cell surface. Moreover, using molecular approaches, the interactions of DUOX with the DUOXA that influence the type of ROS generated (H2O2 or O2.-) are investigated.

 

 

 

 

 

In-vivo investigation of the DUOX/DUOXA physiological roles: Congenital hypothyroidism and transgenic mice models

DX2_mutantsCongenital hypothyroidism (CH) is the most common congenital endocrine disorder affecting 1 in every 3000 newborns. In 20% of the patients, permanent CH is due to biochemical defects causing a dyshormonogenesis. Transient CH is often associated with a temporally limited exposure to external factors during pregnancy such as a lack or an excess of iodide intake, transplacental antibodies, or antithyroid drug-based treatments. A genetic basis for transient CH has been proposed with the discovery of affected patients bearing mutations in the DUOX2 gene. Several published DUOX2 missense mutations have been characterized in the laboratory. We have demonstrated that they cause DUOX expression and/or maturation defects resulting in the absence or reduced H2O2 generation. Recently, we identified the first inactivating DUOX2 mutation localized in the catalytic core of the oxidase. In collaboration with worlwide research units, we continue to screen for natural DUOX/DUOXA mutations in CH patients to help for genetic counseling. In addition, their functional characterization will enable us to better understand their mechanism of maturation and how these new NADPH-oxidases function.

 

  

 

 

 

Mice2

 

In collaboration with several academic groups around the world, we have also access to transgenic “knock-out” mice for DUOX / DUOXA genes. These models will be very useful to analyze the physiological roles of these novel H2O2 generators in the thyroid metabolism, but also in the other tissues expressing the DUOX.

 

 

 

 

 

  

 

 

 

 

 

 

Excess of H2O2: Risk factor in cancer development

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Up to 50% of the population above 60 years old present thyroid nodules and 5% of these nodules will degenerate into cancers. Irradiation is the only environmental risk factor clearly implicated in thyroid cancer pathogenesis. However, irradiation is certainly not responsible for the majority of thyroid tumors. We hypothesized that the elevated frequency of thyroid tumors could be partially explained by the prominent mutagenic environment present in the thyroid, resulting from its metabolism producing large amounts of hydrogen peroxide. We have demonstrated that non-lethal concentrations of H2O2 provoke a large number of mutagenic DNA double strand breaks in primo-cultured human thyrocytes. We have also shown that DNA breaks induced by H2O2 were more slowly repaired than those induced by irradiation supporting the hypothesis that generation of H2O2 in thyroid could also play a role in mutagenesis particularly in case of antioxidant defense deficiency.

  

 

DUOX proteins and H2O2: Role in host defense

TMM1The pseudostratified moist respiratory tract and gastrointestinal mucosae do not form a tightly closed barrier, allowing nutrient exchanges, and exist in transient (respiratory tract, stomach) or permanent (colon) contact with the microflora. DUOX proteins are known to be also expressed at the surface of these highly differentiated epithelia. Multiple functions have been attributed to the DUOX enzymes including innate host defense. This role is believed to be exerted in conjunction with the lactoperoxidase enzyme (LPO) that oxidizes the thiocyanate (SCN-) into the microbicidal compound OSCN-.  However, H2O2 secretion by these mucosae does not occur in the very limited space, undergoing a dilution effect. We have shown that H2O2, under conditions that preserved bacterial growth, exert a repellent effect on Salmonella on agar plates. DUOX transfected cells generating H2O2 in the medium were much less infected by Salmonella than their control counterparts. Normal invasion was restored upon incubation with catalase acting on extracellular but not intracellular H2O2. For its repellent effect, H2O2 does not need to diffuse in high concentrations far from the cells because the prevention of adhesion is sufficient to circumvent microbe infection. This chemorepulsion action of H2O2 would constitute an adequate system to preserve a "peaceful" coexistence between the host and resident saprophytic bacteria, keeping at bay the cells from pathogenic microbes.

 

 

 

Characterization of the thyroid-H2O2 generating system in the zebrafish model

 

Zebrafish has been recognized as an attractive organism to study many biological processes on vertebrate development and the use of zebrafish models has become increasingly popular in various biomedical research topics including the study of molecular mechanisms underlying different human diseases, including cancer. Its high fecundity, rapid development and short generation time make zebrafish a very powerful animal model to perform genetic alterations and large-scale experiments with high reproducibility. The zebrafish thyroid is not organized in one compact gland encapsulated by connective tissue but consist of follicles of variable shape and diameter that lie individually between the first gill arch and the bulbus arteriosus along the ventral aorta. Compared to the two DUOX and two DUOXA genes present in mammals, only one duox and one single duoxa gene have been identified in the zebrafish genome. During thyroid primordium budding and migration, the expression of most of the genes necessary for thyroid hormones synthesis (tshr, tg, tpo, nis, duoxa) appears between 40 and 55 hours post fertilization (hpf). However, the onset of duox expression is delayed appearing at around 80 hpf, corresponding to the beginning of T4 detection. These data suggest that duox function might trigger thyroid hormone synthesis, but its role remains to be investigated in vivo.

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