Missense mutations of dual oxidase 2 (DUOX2) implicated in congenital hypothyroidism have impaired trafficking in cells reconstituted with DUOX2 maturation factor
Missense mutations of dual oxidase 2 (DUOX2) implicated in congenital hypothyroidism have impaired trafficking in cells reconstituted with DUOX2 maturation factor
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DOI：
10.1210/me.2007-0018
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发表时间：
2007-06-01
影响因子：
--
通讯作者：
Refetoff, Samuel
中科院分区：
文献类型：
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作者：
Grasberger, Helmut;De Deken, Xavier;Refetoff, Samuel
双氧化酶2 （DUOX2）是一种还原的NAD磷酸：O-2氧化还原酶黄蛋白，是甲状腺细胞H2O2发生器的一个组成部分，需要在根尖质膜合成激素。我们最近发现了一种特定的DUOX2成熟因子（DUOXA2），它是哺乳动物细胞系中表达功能性DUOX2的必要和充分条件。现在，我们使用DUOXA2重组系统首次在分子水平上表征了天然DUOX2错义变体（Q36H, R376W, D506N），分析了它们对H2O2产生，运输，稳定性，折叠和DUOXA2相互作用的影响。Q36H和R376W突变完全阻止了DUOX2向细胞表面的传递。突变蛋白主要以核心n -糖基化、巯基还原的折叠中间体的形式存在，这些中间体被内质网（ER）的质量控制系统保留，与凝集素钙连联素的络合增加表明了这一点。D506N表现出部分缺陷表型，其表面表达减少的突变蛋白具有正常的产生H2O2的内在活性。D506N n -聚糖部分在高尔基体中不受正常修饰，表明外源蛋白可以逃避内质网的质量控制。内质网中DUOX2的氧化折叠似乎是DUOX2成熟的限速步骤，但DUOXA2并没有促进这一过程。相反，DUOXA2允许折叠后的DUOX2快速退出内质网，或者增强对不能退出内质网的突变体DUOX2蛋白的降解。因此，DUOXA2可能是DUOX2专用的二级质量控制体系的一部分。
Dual oxidase 2 ( DUOX2), a reduced NAD phosphate: O-2 oxidoreductase flavoprotein, is a component of the thyrocyte H2O2 generator required for hormone synthesis at the apical plasma membrane. We recently identified a specific DUOX2 maturation factor (DUOXA2) that is necessary and sufficient for expression of functional DUOX2 in mammalian cell lines. We have now used a DUOXA2 reconstituted system to provide the first characterization of natural DUOX2 missense variants (Q36H, R376W, D506N) at the molecular level, analyzing their impact on H2O2 generation, trafficking, stability, folding, and DUOXA2 interaction. The Q36H and R376W mutations completely prevent routing of DUOX2 to the cell surface. The mutant proteins are predominantly present as core N-glycosylated, thiol-reduced folding intermediates, which are retained by the quality control system within the endoplasmic reticulum (ER) as indicated by increased complexation with the lectin calnexin. D506N displays a partial deficiency phenotype with reduced surface expression of a mutant protein with normal intrinsic activity in generating H2O2. D506N N-glycan moieties are not subject to normal modification in the Golgi apparatus, suggesting that nonnative protein can escape the quality control in the ER. Oxidative folding of DUOX2 in the ER appears to be the rate-limiting step in the maturation of DUOX2, but is not facilitated by DUOXA2. Rather, DUOXA2 allows rapid ER exit of folded DUOX2 or enhanced degradation of mutant DUOX2 proteins not competent for ER exit. DUOXA2 may thus be part of a secondary quality control system specific for DUOX2.