RESUMO
CONTEXT: 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. OBJECTIVE: The objective of this study was to evaluate the functional consequences of TPO-DUOX interaction at the plasma membrane. DESIGN: 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. RESULTS: We observed that production of H2O2 decreases both TPO and DUOX activities. We show that TPO presents a catalase-like effect that protects DUOX from inhibition by H2O2. This catalase-like effect depends on the association between both enzymes, which probably occurs through the DUOX peroxidase-like domain because this effect was not observed with human DUOX2 mutants. CONCLUSION: The DUOX-TPO association at the plasma membrane is relevant for normal enzyme properties. Normally, TPO consumes H2O2 produced by DUOX, decreasing the availability of this substance at the apical membrane of thyrocytes and, in turn, probably decreasing the oxidative damage of macromolecules.
Assuntos
Autoantígenos/metabolismo , Membrana Celular/enzimologia , Iodeto Peroxidase/metabolismo , Proteínas de Ligação ao Ferro/metabolismo , NADPH Oxidases/metabolismo , Oxirredutases/metabolismo , Autoantígenos/genética , Catalase/metabolismo , Oxidases Duais , Citometria de Fluxo , Regulação Enzimológica da Expressão Gênica , Células HEK293 , Humanos , Peróxido de Hidrogênio/metabolismo , Iodeto Peroxidase/genética , Proteínas de Ligação ao Ferro/genética , Rim/enzimologia , NADPH Oxidases/genética , Oligonucleotídeos Antissenso , TransfecçãoRESUMO
During childhood, the thyroid gland is one of the most sensitive organs to the carcinogenetic effects of ionizing radiation that may lead to papillary thyroid carcinoma (PTC) associated with RET/PTC oncogene rearrangement. Exposure to ionizing radiation induces a transient "oxidative burst" through radiolysis of water, which can cause DNA damage and mediates part of the radiation effects. H(2)O(2) is a potent DNA-damaging agent that induces DNA double-strand breaks, and consequently, chromosomal aberrations. Irradiation by 5 Gy X-ray increased extracellular H(2)O(2). Therefore, we investigated the implication of H(2)O(2) in the generation of RET/PTC1 rearrangement after X-ray exposure. We developed a highly specific and sensitive nested reverse transcription-PCR method. By using the human thyroid cell line HTori-3, previously found to produce RET/PTC1 after gamma-irradiation, we showed that H(2)O(2), generated during a 5 Gy X-ray irradiation, causes DNA double-strand breaks and contributes to RET/PTC1 formation. Pretreatment of cells with catalase, a scavenger of H(2)O(2), significantly decreased RET/PTC1 rearrangement formation. Finally, RET/PTC chromosomal rearrangement was detected in HTori-3.1 cells after exposure of cells to H(2)O(2) (25 micromol/L), at a dose that did not affect the cell viability. This study shows for the first time that H(2)O(2) is able to cause RET/PTC1 rearrangement in thyroid cells and consequently highlights that oxidative stress could be responsible for the occurrence of RET/PTC1 rearrangement found in thyroid lesions even in the absence of radiation exposure.