Corinne Dupuy - Academia.edu (original) (raw)

Papers by Corinne Dupuy

Anti Cancer Agents in Medicinal Chemistry, Aug 27, 2012

Journal of Biological Chemistry

The thyroid plasma membrane contains a Ca2(+)-regulated NADPH-dependent H2O2 generating system wh... 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-.

Applied Physics Letters, 2015

ABSTRACT Phase images of biological specimens were obtained by the method of Quadriwave Lateral S... more ABSTRACT Phase images of biological specimens were obtained by the method of Quadriwave Lateral Shearing Interferometry (QWLSI). The QWLSI technique produces, at high resolution, phase images of the cells having been exposed to a plasma treatment and enables the quantitative analysis of the changes in the surface area of the cells over time. Morphological changes in the HTori normal thyroid cells were demonstrated using this method. There was a comparison of the cell behaviour between control cells, cells treated by plasma of a nanosecond dielectric barrier discharge, including cells pre-treated by catalase, and cells treated with an equivalent amount of H2O2. The major changes in the cell membrane morphology were observed at only 5 min after the plasma treatment. The primary role of reactive oxygen species (ROS) in this degradation is suggested. Deformation and condensation of the cell nucleus were observed 2–3 h after the treatment and are supposedly related to apoptosis induction. The coupling of the phase QWLSI with immunofluorescence imaging would give a deeper insight into the mechanisms of plasma induced cell death.

Polymer Engineering & Science, 2003

Proceedings of the National Academy of Sciences of the United States of America, Jan 6, 2015

Ionizing radiation (IR) causes not only acute tissue damage, but also late effects in several cel... more Ionizing radiation (IR) causes not only acute tissue damage, but also late effects in several cell generations after the initial exposure. The thyroid gland is one of the most sensitive organs to the carcinogenic effects of IR, and we have recently highlighted that an oxidative stress is responsible for the chromosomal rearrangements found in radio-induced papillary thyroid carcinoma. Using both a human thyroid cell line and primary thyrocytes, we investigated the mechanism by which IR induces the generation of reactive oxygen species (ROS) several days after irradiation. We focused on NADPH oxidases, which are specialized ROS-generating enzymes known as NOX/DUOX. Our results show that IR induces delayed NADPH oxidase DUOX1-dependent H2O2 production in a dose-dependent manner, which is sustained for several days. We report that p38 MAPK, activated after IR, increased DUOX1 via IL-13 expression, leading to persistent DNA damage and growth arrest. Pretreatment of cells with catalase, ...

Antioxidants & redox signaling, Jan 11, 2015

The dual oxidase 2 (DUOX2) protein belongs to the NADPH oxidase family. As H2O2 generator it play... more The dual oxidase 2 (DUOX2) protein belongs to the NADPH oxidase family. As H2O2 generator it plays a key role in both thyroid hormone biosynthesis and innate immunity. DUOX2 forms with its maturation factor DUOXA2 a stable complex at the cell surface that is crucial for the H2O2 generating activity but the nature of their interaction is unknown. The contribution of some cysteine residues located in the N-terminal ectodomain of DUOX2 in a surface protein-protein interaction is suggested. We have investigated the involvement of different cysteine residues to the formation of covalent bonds that could be of critical importance for the function of the complex. We report the identification and the characterization of an intramolecular disulfide bond between cys-124 of the N-terminal ectodomain and cys-1162 of an extracellular loop of DUOX2, which has important functional implications in both export and activity of DUOX2. This intramolecular bridge provides structural support for the form...

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Cancer radiothérapie : journal de la Société française de radiothérapie oncologique, 2011

Exposure to ionising radiations during childhood increases the risk of thyroid cancer. Similar ri... more Exposure to ionising radiations during childhood increases the risk of thyroid cancer. Similar risk factors have been found after external radiation exposure or internal contamination with radioactive iodine isotopes. In case of contamination with radioiodines, administration of potassium iodide can prevent thyroid irradiation.

The Biochemical journal, Jan 15, 1997

Pig thyroid plasma membranes contain a Ca(2+)-dependent NADPH:O2 oxidoreductase, the thyroid NADP... more Pig thyroid plasma membranes contain a Ca(2+)-dependent NADPH:O2 oxidoreductase, the thyroid NADPH-dependent H2O2 generator. This provided the H2O2 for the peroxidase-catalysed synthesis of thyroid hormones. The effect of the tervalent arsenical, phenylarsine oxide (PAO), on the NADPH oxidase was studied. PAO caused two directly related dose-dependent effects with similar half-effect concentrations of PAO (3 nmol of PAO/mg of protein): (i) partial inactivation of H2O2 formation by the Ca(2+)-stimulated enzyme, and (ii) desensitization of the enzyme activity to Ca2+. PAO had no effect on membranes that had been Ca(2+)-desensitized by alpha-chymotrypsin treatment. The NADPH oxidase in membranes treated with excess PAO had the same Vmax with and without Ca2+. This value was half the Vmax of the native enzyme. However, the K(m) for NADPH determined with Ca2+ (18 microM, identical with that of the native enzyme) was approx, one-third of the K(m) measured without Ca2+, showing the direct ...

The Biochemical journal, 1994

The thyroid plasma membrane contains a Ca(2+)-regulated NADPH-dependent H2O2-generating system wh... more The thyroid plasma membrane contains a Ca(2+)-regulated NADPH-dependent H2O2-generating system which provides H2O2 for the peroxidase-catalysed biosynthesis of thyroid hormones. The electron transfer from NADPH to O2 catalysed by this system was studied by using diphenyleneiodonium (DPI), an inhibitor of flavo- and haemo-proteins. The prosthetic group of the H2O2 generator was removed by incubation with 5 mM CHAPS at 40 degrees C, and an active holoenzyme was reconstituted with FAD, but not with FMN. The H2O2-generating system also had an intrinsic Ca(2+)-dependent NADPH:ferricyanide reductase activity which is probably linked to its flavodehydrogenase component (or domain). Both activities, H2O2 production and ferricyanide reductase activity, were inhibited by DPI, with similar K1/2 (2.5 nmol/mg of protein). DPI only inhibited a system reduced with NADPH in the presence of Ca2+. NADPH could not be replaced by NADP+, NADH or sodium dithionite, suggesting the need for specific mild r...

$Research paper thumbnail of Activation of the NADPH-dependent H2O2-generating system in pig thyroid particulate fraction by limited proteolysis and Zn2+ treatment$

The Biochemical journal, Jan 15, 1992

The NADPH-dependent H2O2-generating system in a pig thyroid particulate fraction requires micromo... more The NADPH-dependent H2O2-generating system in a pig thyroid particulate fraction requires micromolar concentrations of Ca2+ for activity. The H2O2 generator could be Ca(2+)-desensitized (i.e. made fully active in the absence of Ca2+) by limited proteolysis with alpha-chymotrypsin or by treatment with ZnCl2. The Zn2+ effect was temperature- and dose-dependent with an apparent half-maximum concentration of 0.15 mM at 40 degrees C. Ca2+ desensitization was not reversed by adding the Zn2+ chelators, 1,10-phenanthroline and EGTA, but about one-third of the Ca(2+)-sensitivity was recovered after addition of 10 mM-dithiothreitol. The proteolysed enzyme and the Zn(2+)-treated enzyme had different Km values for NADPH. The Zn2+ effect did not seem to involve proteolysis or membrane fusion. These results indicate that Ca2+ regulation occurs via an autoinhibitory domain or inhibitory protein component of the H2O2-generator system. Its inhibitory effect may be removed by proteolysis or conformat...

Thyroid : official journal of the American Thyroid Association, 2006

The human iodotyrosine dehalogenase 1 (DEHAL1) gene is composed of six exons. Two isoforms (DEHAL... more The human iodotyrosine dehalogenase 1 (DEHAL1) gene is composed of six exons. Two isoforms (DEHAL1 and DEHAL1B) have been published in GenBank, both of which have a nitroreductase domain and arise from differential splicing in exon 5. We recently showed that the DEHAL1 isoform is a transmembrane protein that efficiently catalyzes the NADPH-dependent deiodination of mono (L-MIT) and diiodotyrosine (L-DIT) in human embryonic kidney-293 (HEK293) cells. In the present study, we establish the existence of a new transcript, DEHAL1C, in the human thyroid with a terminal exon that lacks in the DEHAL1 transcript. This exon is the complete exon 5, which is spliced in the DEHAL1B mRNA variant. These two variants encode proteins with differing C-terminal domains. Using quantitative reverse transcription polymerase chain reaction, we found that the expression of the mRNA of DEHAL1C and DEHAL1B was lower than that of DEHAL1 mRNA in the thyroid. We also observed that human DEHAL1B and DEHAL1C prot...

The Journal of clinical endocrinology and metabolism, 2010

Thyroperoxidase (TPO) and dual oxidase (DUOX) are present at the apical membrane of thyrocytes, w... more Thyroperoxidase (TPO) and dual oxidase (DUOX) are present at the apical membrane of thyrocytes, where TPO catalyzes thyroid hormone biosynthesis in the presence of H2O2 produced by DUOX. Both enzymes are colocalized and associated, but the consequences of this interaction remain obscure. The objective of this study was to evaluate the functional consequences of TPO-DUOX interaction at the plasma membrane. The functional consequences of DUOX-TPO interaction were studied by measuring extracellular H2O2 concentration and TPO activity in a heterologous system. For this purpose, HEK293 cells were transiently transfected with a combination of human TPO with human DUOX1 or DUOX2 in the presence of their respective maturation factors, DUOXA1 or DUOXA2. The effect of human DUOX2 mutants in which cysteine residues in the N-terminal domain were replaced by glycines was also analyzed. We observed that production of H2O2 decreases both TPO and DUOX activities. We show that TPO presents a catalas...

European Thyroid Journal, 2013

ticipation of NADPH oxidases not only in thyroid physiology but also in gland pathophysiology, pa... more ticipation of NADPH oxidases not only in thyroid physiology but also in gland pathophysiology, particularly the involvement of these enzymes in the regulation of thyroid oxidative stress.

The Journal of biological chemistry, Jan 6, 2015

NADPH oxidase (Nox) family proteins produce superoxide (O2 (⨪)) directly by transferring an elect... more 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 (⨪) re...

ISRN Inflammation, 2012

In cystic fibrosis (CF) patients, pulmonary inflammation is a major cause of morbidity and mortal... more In cystic fibrosis (CF) patients, pulmonary inflammation is a major cause of morbidity and mortality. The aim of this study was to further investigate whether oxidative stress could be involved in the early inflammatory process associated with CF pathogenesis. We used a model of CFTR defective epithelial cell line (IB3-1) and its reconstituted CFTR control (S9) cell line cultured in various ionic conditions. This study showed that IB3-1 and S9 cells expressed the NADPH oxidases (NOXs) DUOX1/2 and NOX2 at the same level. Nevertheless, several parameters participating in oxidative stress (increased ROS production and apoptosis, decreased total thiol content) were observed in IB3-1 cells cultured in hypertonic environment as compared to S9 cells and were inhibited by diphenyleneiodonium (DPI), a well-known inhibitor of NOXs; besides, increased production of the proinflammatory cytokines IL-6 and IL-8 by IB3-1 cells was also inhibited by DPI as compared to S9 cells. Furthermore, calcium ionophore (A23187), which upregulates DUOX and NOX2 activities, strongly induced oxidative stress and IL-8 and IL-6 overexpression in IB3-1 cells. All these events were suppressed by DPI, supporting the involvement of NOXs in the oxidative stress, which can upregulate proinflammatory cytokine production by the airway CFTR-deficient cells and trigger early pulmonary inflammation in CF patients.

Thyroid, 2013

AU3 c Background: Dual oxidases (DUOX1 and DUOX2) are NADPH oxidases (NOX) involved in hydrogen p... more AU3 c Background: Dual oxidases (DUOX1 and DUOX2) are NADPH oxidases (NOX) involved in hydrogen peroxide production necessary for thyroid hormonogenesis, but recently, the NOX4 has also been described in the thyroid gland. The prevalence of thyroid disease is higher in women, and the basis for this difference might involve a higher oxidative stress level in the female thyroid gland. Hence, we aimed at evaluating whether the function and the expression of enzymes involved in the thyroid redox balance differ between females and males. Methods: DUOX1, DUOX2, NOX4, glutathione peroxidase (GPx), and catalase activities and expression levels were evaluated in the thyroids of prepubertal and adult male and female rats. The mRNA levels of DUOXA1 and DUOXA2, the DUOX maturation factors, and of p22phox and Poldip2 (subunits of NOX4) were also determined.

European Journal of Biochemistry, 1996

The thyroid plasma membrane contains a Ca"-regulated NADPH-dependent H,O,-generating system which... more The thyroid plasma membrane contains a Ca"-regulated NADPH-dependent H,O,-generating system which provides H,O, for the thyroid-peroxidase-catalyzed biosynthesis of thyroid hormones. The molecular nature of the membrane-associated electron transport chain that generates H,O, in the thyroid is unknown, but recent observations indicate that a flavoprotein containing a FAD prosthetic group is involved. Solubilization was reinvestigated using zyxwvu 3-[(3-cholamidopropyl)dimethylammonio]-l -propanesulfonate (Chaps), Triton X-100, and high salt concentrations. Chaps eliminated about 30% of the proteins, which included a ferricyanide reductase, without affecting the H,O,-generating system. Similarly, Triton X-100 alone did not extract the NADPH oxidase. An NADPH-oxidase activity, which was measured in the presence of the artificial electron acceptor potassium ferricyanide, was solubilized by increasing the ionic strength to 2 M KCI. This NADPH-ferricyanide reductase activity was shown to belong to the H,O,generating system, although it did not produce H,O,. It was still Ca*+ dependent and H,O, production was restored by decreasing the ionic strength by overnight dialysis. No H,O, production activity was detected after sucrose density gradient centrifugation of the dialyzed solubilized enzyme, but a welldefined peak of NADPH oxidation activity with a sedimentation coefficient of 3.71 S was found in the presence of K,Fe(CN),. These results suggest that some unknown component(s) (phospholipid or protein) is removed during sucrose density gradient centrifugation. Finally, thyrotropin, which induces NADPH oxidase and regulates H,O, production in porcine thyrocytes in primary culture, also induced the NADPH-K,Fe(CN), reductase activity associated with the H,O,-generating system. Thus, this enzyme seems to be another marker of thyroid differentiation.

Thyroid, 2014

Homozygous loss-of-function mutations in the FOXE1 gene have been reported in several patients wi... more Homozygous loss-of-function mutations in the FOXE1 gene have been reported in several patients with partial or complete Bamforth-Lazarus syndrome: congenital hypothyroidism (CH) with thyroid dysgenesis (usually athyreosis), cleft palate, spiky hair, with or without choanal atresia, and bifid epiglottis. Here, our objective was to evaluate potential functional consequences of a FOXE1 mutation in a patient with a similar clinical phenotype. FOXE1 was sequenced in eight patients with thyroid dysgenesis and cleft palate. Transient transfection was performed in HEK293 cells using the thyroglobulin (TG) and thyroid peroxidase (TPO) promoters in luciferase reporter plasmids to assess the functional impact of the FOXE1 mutations. Primary human thyrocytes transfected with wild type and mutant FOXE1 served to assess the impact of the mutation on endogenous TG and TPO expression. We identified and characterized the function of a new homozygous FOXE1 missense mutation (p.R73S) in a boy with a typical phenotype (athyreosis, cleft palate, and partial choanal atresia). This new mutation located within the forkhead domain was inherited from the heterozygous healthy consanguineous parents. In vitro functional studies in HEK293 cells showed that this mutant gene enhanced the activity of the TG and TPO gene promoters (1.5-fold and 1.7-fold respectively vs. wild type FOXE1; p&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;0.05), unlike the five mutations previously reported in Bamforth-Lazarus syndrome. The gain-of-function effect of the FOXE1-p.R73S mutant gene was confirmed by an increase in endogenous TG production in primary human thyrocytes. We identified a new homozygous FOXE1 mutation responsible for enhanced expression of the TG and TPO genes in a boy whose phenotype is similar to that reported previously in patients with loss-of-function FOXE1 mutations. This finding further delineates the role for FOXE1 in both thyroid and palate development, and shows that enhanced gene activity should be considered among the mechanisms underlying Bamforth-Lazarus syndrome.