Donkó Ágnes, Morand Stanislas, Korzeniowska Agnieszka, Boudreau Howard E, Zana Melinda, Hunyady László, Geiszt Miklós, Leto Thomas L
Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD 20852, USA; Department of Physiology, Semmelweis University, Budapest, Hungary; "Lendület" Peroxidase Enzyme Research Group of the Semmelweis University and the Hungarian Academy of Sciences, Budapest, Hungary.
Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD 20852, USA.
Free Radic Biol Med. 2014 Aug;73:190-200. doi: 10.1016/j.freeradbiomed.2014.05.006. Epub 2014 May 20.
In the thyroid gland Duox2-derived H2O2 is essential for thyroid hormone biosynthesis. Several patients were identified with partial or severe iodide organification defects caused by mutation in the gene for Duox2 or its maturation factor, DuoxA2. A Duox2-deficient (Duox2(thyd)) mouse model enabled in vivo investigation of its critical function in thyroid tissues, but its roles proposed in host defense or other innate responses in nonthyroid tissues remain less certain. These mice carry a spontaneous DUOX2 missense mutation, a T→G transversion, in exon 16 that changes the highly conserved valine 674 to glycine and results in severe congenital hypothyroidism. The exact mechanism underlying the effects of the V674G mutation has not been elucidated at the molecular or cellular level. To determine how the V674G mutation leads to congenital hypothyroidism, we introduced the same mutation into human Duox2 or Duox1 cDNAs and expressed them in HEK-293 cells stably expressing the corresponding DuoxA proteins. We found that the valine→glycine mutant Duox proteins fail to produce H2O2, lose their plasma membrane localization pattern, and are retained within the endoplasmic reticulum. The Duox2 mutant binds to DuoxA2, but appears to be unstable owing to this retention. Immunohistochemical staining of Duox2 in murine salivary gland ducts showed that Duox2 in mutant mice loses its condensed apical plasma membrane localization pattern characteristic of wild-type Duox2 and accumulates in punctate vesicular structures within cells. Our findings demonstrate that changing the highly conserved valine 674 in Duox2 leads to impaired subcellular targeting and reactive oxygen species release required for hormonogenesis, resulting in congenital hypothyroidism.
在甲状腺中，双氧化酶2（Duox2）产生的过氧化氢（H2O2）对甲状腺激素的生物合成至关重要。已鉴定出数名患者因Duox2基因或其成熟因子DuoxA2发生突变而出现部分或严重的碘有机化缺陷。一种Duox2缺陷（Duox2(thyd)）小鼠模型能够在体内研究其在甲状腺组织中的关键功能，但其在非甲状腺组织的宿主防御或其他先天反应中所起的作用仍不太明确。这些小鼠在第16外显子中携带一个自发的DUOX2错义突变，即T→G颠换，该突变将高度保守的缬氨酸674变为甘氨酸，导致严重的先天性甲状腺功能减退。V674G突变效应的具体分子或细胞机制尚未阐明。为了确定V674G突变如何导致先天性甲状腺功能减退，我们将相同的突变引入人Duox2或Duox1的cDNA中，并在稳定表达相应DuoxA蛋白的HEK - 293细胞中进行表达。我们发现缬氨酸→甘氨酸突变的Duox蛋白无法产生H2O2，失去其质膜定位模式，并滞留在内质网中。Duox2突变体与DuoxA2结合，但由于这种滞留似乎不稳定。对小鼠唾液腺导管中Duox2的免疫组织化学染色显示，突变小鼠中的Duox2失去了野生型Duox2特有的浓缩顶端质膜定位模式，并在细胞内的点状囊泡结构中积累。我们的研究结果表明，改变Duox2中高度保守的缬氨酸674会导致激素生成所需的亚细胞靶向和活性氧释放受损，从而导致先天性甲状腺功能减退。
