Publications

• Biochemical characterization of a Ca2+/NAD(P)H-dependent H2O2 generator in human thyroid tissue.

  Leseney A.M., Deme D., Legue O., Ohayon R., Chanson P., Sales J.P., Carvalho D.P., Dupuy C., Virion A.

  An NAD(P)H-dependent H2O2 forming activity has been evidenced in thyroid tissue from patients with Grave's disease. Its biochemical properties were compared to those of the NADPH oxidase previously described in pig thyroid gland. Both were Ca2+-dependent and activated by inorganic phosphate anions ... >> More

  An NAD(P)H-dependent H2O2 forming activity has been evidenced in thyroid tissue from patients with Grave's disease. Its biochemical properties were compared to those of the NADPH oxidase previously described in pig thyroid gland. Both were Ca2+-dependent and activated by inorganic phosphate anions in the same range of concentrations. Both are flavoproteins using FAD as cofactor, but the human enzyme was also able to utilize FMN. The apparent Km for NADPH of the human enzyme (100 microM) was 5-10 times higher than that of porcine enzyme. Vm was 3 to 10 times higher in pig (150 nmol x h(-1) x mg(-1)) than in man (14 to 45). Total content in human tissue was 7 to 9% of that in porcine tissue. An unidentified inhibitor has been detected in the 3000 g particulate fraction from most patients, which could account for this apparently low enzyme content. An NADH-dependent H2O2 production has also been observed in porcine and human thyroid tissues. This activity was only partly Ca2+-dependent (man, 50-70%; pig, 80-90%) and presented similar apparent Km values for NADH (man, 100 microM; pig, 200 microM). In pig thyrocytes, the expression of the Ca2+-dependent part of the NADH-oxidase activity was induced by TSH and down-regulated by TGFbeta, as was the NADPH oxidase activity. Furthermore, NADPH and NADH-dependent activities were not additive. We conclude that a single, inducible, NAD(P)H-oxidase can use NADPH or NADH as substrate to catalyse H2O2 formation, and that human and porcine NAD(P)H-oxidases are highly similar. Differences observed could be attributed to minor differences in enzyme structure and/or in membrane microenvironment. The NADH-dependent Ca2+-independent activity observed in human and porcine thyroid fractions could be attributed to a distinct and constitutive enzyme. << Less

  Biochimie 81:373-380(1999) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Purification of a novel flavoprotein involved in the thyroid NADPH oxidase. Cloning of the porcine and human cDNAs.

  Dupuy C., Ohayon R., Valent A., Noel-Hudson M.-S., Deme D., Virion A.

  Hydrogen peroxide is the final electron acceptor for the biosynthesis of thyroid hormone catalyzed by thyroperoxidase at the apical surface of thyrocytes. Pig and human thyroid plasma membrane contain a Ca(2+)-dependent NAD(P)H oxidase that generates H(2)O(2) by transferring electrons from NAD(P)H ... >> More

  Hydrogen peroxide is the final electron acceptor for the biosynthesis of thyroid hormone catalyzed by thyroperoxidase at the apical surface of thyrocytes. Pig and human thyroid plasma membrane contain a Ca(2+)-dependent NAD(P)H oxidase that generates H(2)O(2) by transferring electrons from NAD(P)H to molecular oxygen. We purified from pig thyroid plasma membrane a flavoprotein which constitutes the main, if not the sole, component of the thyroid NAD(P)H oxidase. Microsequences permitted the cloning of porcine and human full-length cDNAs encoding, respectively, 1207- and 1210-amino acid proteins with a predicted molecular mass of 138 kDa (p138(Tox)). Human and porcine p138(Tox) have 86.7% identity. The strongest similarity was to a predicted polypeptide encoded by a Caenorhabditis cDNA and with rbohA, a protein involved in the Arabidopsis NADPH oxidase. p138(Tox) shows also similarity to the p65(Mox) and to the gp91(Phox) in their C-terminal region and have consensus sequences for FAD- and NADPH-binding sites. Compared with gp91(Phox), p138(Tox) shows an extended N-terminal containing two EF-hand motifs that may account for its calcium-dependent activity, whereas three of four sequences implicated in the interaction of gp91(Phox) with the p47(Phox) cytosolic factor are absent in p138(Tox). The expression of porcine p138(Tox) mRNA analyzed by Northern blot is specific of thyroid tissue and induced by cyclic AMP showing that p138(Tox) is a differentiation marker of thyrocytes. The gene of human p138(Tox) has been localized on chromosome 15q15. << Less

  J. Biol. Chem. 274:37265-37269(1999) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Mechanism of hydrogen peroxide formation catalyzed by NADPH oxidase in thyroid plasma membrane.

  Dupuy C., Virion A., Ohayon R., Kaniewski J., Deme D., Pommier J.

  The thyroid plasma membrane contains a Ca2(+)-regulated NADPH-dependent H2O2 generating system which provides H2O2 for the thyroid peroxidase-catalyzed biosynthesis of thyroid hormones. The plasma membrane fraction contains a Ca2(+)-independent cytochrome c reductase activity which is not inhibite ... >> More

  The thyroid plasma membrane contains a Ca2(+)-regulated NADPH-dependent H2O2 generating system which provides H2O2 for the thyroid peroxidase-catalyzed biosynthesis of thyroid hormones. The plasma membrane fraction contains a Ca2(+)-independent cytochrome c reductase activity which is not inhibited by superoxide dismutase. But it is not known whether H2O2 is produced directly from molecular oxygen (O2) or formed via dismutation of super-oxide anion (O2-). Indirect evidence from electron scavenger studies indicate that the H2O2 generating system does not liberate O2-, but studies using the modified peroxidase, diacetyldeuteroheme horseradish peroxidase, to detect O2-indicate that H2O2 is provided via the dismutation of O2-. The present results provide indirect evidence that the cytochrome c reductase activity is not a component of the NADPH-dependent H2O2 generator, since it was removed by washing the plasma membranes with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid without affecting H2O2 generation. Spectral studies with diacetyldeuteroheme-substituted horseradish peroxidase showed that the thyroid NADPH-dependent H2O2 generator does not catalyze superoxide anion formation. The O2-adduct compound (compound III) was formed but was completely inhibited by catalase, indicating that the initial product was H2O2. The rate of NADPH oxidation also increased in the presence of diacetylheme peroxidase. This increase was blocked by catalase and was greatly enhanced by superoxide dismutase. The O2-adduct compound (compound III) was produced in the presence of NADPH when glucose-glucose oxidase (which does not produce O2-) was used as the H2O2 generator. NADPH oxidation occurred simultaneously and was enhanced by superoxide dismutase. We conclude that O2-formation occurs in the presence of an H2O2 generator, diacetylheme peroxidase and NADPH, but that it is not the primary product of the H2O2 generator. We suggest that O2-formation results from oxidation of NADPH, catalyzed by the diacetylheme peroxidase compound I, producing NADP degree, which in turn reacts with O2 to give O2-. << Less

  J Biol Chem 266:3739-3743(1991) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.

• Inactivating mutations in the gene for thyroid oxidase 2 (THOX2) and congenital hypothyroidism.

  Moreno J.C., Bikker H., Kempers M.J.E., van Trotsenburg A.S., Baas F., de Vijlder J.J.M., Vulsma T., Ris-Stalpers C.

  <h4>Background</h4>Several genetic defects are associated with permanent congenital hypothyroidism. Immunologic, environmental, and iatrogenic (but not genetic) factors are known to induce transient congenital hypothyroidism, which spontaneously resolves within the first months of life. We hypothe ... >> More

  <h4>Background</h4>Several genetic defects are associated with permanent congenital hypothyroidism. Immunologic, environmental, and iatrogenic (but not genetic) factors are known to induce transient congenital hypothyroidism, which spontaneously resolves within the first months of life. We hypothesized that molecular defects in the thyroid oxidase system, which is composed of at least two proteins, might be involved in the pathogenesis of permanent or transient congenital hypothyroidism in babies with defects in iodide organification, for which the oxidase system is required.<h4>Methods</h4>Nine patients were recruited who had idiopathic congenital hypothyroidism (one with permanent and eight with transient hypothyroidism) and an iodide-organification defect and who had been identified by the screening program for congenital hypothyroidism. The DNA of the patients and their relatives was analyzed for mutations in the genes for thyroid oxidase 1 (THOX1 ) and 2 (THOX2 ).<h4>Results</h4>The one patient with permanent and severe thyroid hormone deficiency and a complete iodide-organification defect had a homozygous nonsense mutation in the THOX2 gene that eliminates all functional domains of the protein. Three of the eight patients with mild transient congenital hypothyroidism and a partial iodide-organification defect had heterozygous mutations in the THOX2 gene that prematurely truncate the protein, thus abolishing its functional domains.<h4>Conclusions</h4>Biallelic inactivating mutations in the THOX2 gene result in complete disruption of thyroid-hormone synthesis and are associated with severe and permanent congenital hypothyroidism. Monoallelic mutations are associated with milder, transient hypothyroidism caused by insufficient thyroidal production of hydrogen peroxide, which prevents the synthesis of sufficient quantities of thyroid hormones to meet the large requirement for thyroid hormones at the beginning of life. << Less

  N. Engl. J. Med. 347:95-102(2002) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Cloning of two human thyroid cDNAs encoding new members of the NADPH oxidase family.

  De Deken X., Wang D., Many M.-C., Costagliola S., Libert F., Vassart G., Dumont J.E., Miot F.

  Two cDNAs encoding NADPH oxidases and constituting the thyroid H(2)O(2) generating system have been cloned. The strategy of cloning was based on the functional similarities between H(2)O(2) generation in leukocytes and the thyroid, according to the hypothesis that one of the components of the thyr ... >> More

  Two cDNAs encoding NADPH oxidases and constituting the thyroid H(2)O(2) generating system have been cloned. The strategy of cloning was based on the functional similarities between H(2)O(2) generation in leukocytes and the thyroid, according to the hypothesis that one of the components of the thyroid system would belong to the gp91(Phox)/Mox1 gene family and display sequence similarities with gp91(Phox). Screening at low stringency with a gp91(Phox) probe of cDNA libraries from thyroid cells in primary culture yielded two distinct human cDNA clones harboring open reading frames of 1551 (ThOX1) and 1548 amino acids (ThOX2), respectively. The encoded polypeptides display 83% sequence similarity and are clearly related to gp91(Phox) (53 and 47% similarity). The theoretical molecular mass of 177 kDa is close to the apparent molecular mass of 180 kDa of the native corresponding porcine flavoprotein and the protein(s) detected by Western blot in dog and human thyroid. ThOX1 and ThOX2 display sequence similarities of 53% and 61%, respectively, with a predicted protein of Caenorhabditis elegans over their entire length. They show along their first 500 amino acids a similarity of 43% with thyroperoxidase. The corresponding genes of ThOX1 and ThOX2 are closely linked on chromosome 15q15.3. The dog mRNA expression is thyroid-specific and up-regulated by agents activating the cAMP pathway as is the synthesis of the polypeptides they are coding for. In human thyroid the positive regulation by cAMP is less pronounced. The proteins ThOX1 and ThOX2 accumulate at the apical membrane of thyrocytes and are co-localized with thyroperoxidase. << Less

  J. Biol. Chem. 275:23227-23233(2000) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Characterization of ThOX proteins as components of the thyroid H(2)O(2)-generating system.

  De Deken X., Wang D., Dumont J.E., Miot F.

  We have recently cloned two thyroid-specific cDNAs encoding new members of the NADPH oxidase family. ThOX1 and ThOX2 proteins are colocalized with thyroperoxidase at the apical membrane of human thyroid cells. In the present study we have determined their subcellular localization and maturation in ... >> More

  We have recently cloned two thyroid-specific cDNAs encoding new members of the NADPH oxidase family. ThOX1 and ThOX2 proteins are colocalized with thyroperoxidase at the apical membrane of human thyroid cells. In the present study we have determined their subcellular localization and maturation in relation to their enzymatic activity. A majority of ThOX proteins accumulated inside the cell and only a small fraction was expressed at the surface. Western blots demonstrated that ThOX's are glycoproteins of 180,000 and 190,000. When totally deglycosylated the molecular weight of both ThOX1 and ThOX2 drops to 160,000. Ca(2+) stimulates the basal H(2)O(2) generation in PC Cl3 cells at a level corresponding to 20% of the leukocyte H(2)O(2) production stimulated by PMA. Nonthyroid cell lines transfected with ThOX1 and ThOX2 show only a single immunoreactive band in Western blot analysis, corresponding to the protein of 180,000. This "immature" protein remains exclusively intracellular and does not present any enzymatic activity. This is not modified by coexpression of thyroperoxidase and p22(Phox). Transfection of ThOX cDNAs into PLB-XCGD cells does not reconstitute their NADPH oxidase activity. We conclude that (1) the thyroid contains some elements of the leukocyte H(2)O(2)-generating system but not all of them; (2) ThOX's are predominantly or exclusively located inside the cell in thyrocytes or in transfected cells, respectively, and as such they are inactive; (3) ThOX's cannot replace gp91(Phox) in the leukocyte; and (4) the thyroid H(2)O(2)-generating system is analogous to the leukocyte system with regard to ThOX's and gp91(Phox) but very different in other aspects. Additional thyroid-specific components are probably required to get complete protein processing and full enzymatic activity in the thyroid. << Less

  Exp. Cell Res. 273:187-196(2002) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.