Laissue Paul, Copelli Silvia, Bergada Ignacio, Bergada Cesar, Barrio Gabriel, Karaboga Sinan, Wurtz Jean Marie, Fellous Marc, Lalli Enzo, Veitia Reiner A
U567 INSERM and Université Denis Diderot/Paris VII, Paris, France.
Clin Endocrinol (Oxf). 2006 Nov;65(5):681-6. doi: 10.1111/j.1365-2265.2006.02649.x.
Mutations in DAX-1, an X-linked gene encoding an orphan nuclear receptor, have been associated with adrenal hypoplasia congenita and hypogonadotropic hypogonadism. Here we describe two novel DAX-1 mutations, Y214X and I361T, associated with childhood-onset primary adrenal failure. We aimed at analysing their effects on protein localization, transcriptional activity and propose a structural-function relationship.
We have directly sequenced the DAX-1 gene from PCR-amplified DNA. The effect of the mutations on protein localization was assessed by immunocytochemistry in transfected cells. The impact of mutations on transcriptional activity was determined by transfection using a StAR promoter-luciferase reporter system.
The mutation Y214X produces a protein lacking the C-terminal half of DAX-1. The other one, I361T, affects a highly conserved amino acid within the putative ligand-binding domain. The mutant Y214X displayed a wild-type subcellular localization while I361T was partially retained in the cytoplasm. Both mutants retained subtantial transcriptional repressive activity, compared to mutants producing early onset adrenal failure. Interestingly, I361T displayed similar in vitro transcriptional repressive activity to the mutant I439S previously described in a case of late-onset AHC. This shows that molecular alterations of DAX-1 leading to similar in vitro activities may result in very different ages of onset of adrenal failure, which suggests that additional genetic and epigenetic factors are important in determining the clinical course of AHC.
Our results help the understanding of structure-function relationships in the DAX-1 molecule, suggesting that the N-terminal domain is relatively autonomous and add credence to presumed molecular interactions within ligand binding domain of the protein.
DAX-1是一种编码孤儿核受体的X连锁基因，其突变与先天性肾上腺发育不全和低促性腺激素性性腺功能减退有关。在此，我们描述了两个与儿童期原发性肾上腺功能衰竭相关的新型DAX-1突变，即Y214X和I361T。我们旨在分析它们对蛋白质定位、转录活性的影响，并提出结构-功能关系。
我们对PCR扩增的DNA直接进行DAX-1基因测序。通过转染细胞中的免疫细胞化学评估突变对蛋白质定位的影响。使用类固醇生成急性调节蛋白（StAR）启动子-荧光素酶报告系统通过转染确定突变对转录活性的影响。
Y214X突变产生一种缺乏DAX-1 C端一半的蛋白质。另一个突变I361T影响假定配体结合域内一个高度保守的氨基酸。Y214X突变体表现出野生型亚细胞定位，而I361T部分保留在细胞质中。与导致早发性肾上腺功能衰竭的突变体相比，这两个突变体均保留了相当程度的转录抑制活性。有趣的是，I361T在体外表现出与先前在一例迟发性先天性肾上腺发育不全（AHC）中描述的I439S突变体相似的转录抑制活性。这表明导致相似体外活性的DAX-1分子改变可能导致肾上腺功能衰竭的发病年龄非常不同，这表明额外的遗传和表观遗传因素在决定AHC的临床病程中很重要。
我们的结果有助于理解DAX-1分子中的结构-功能关系，表明N端结构域相对自主，并进一步证明了该蛋白配体结合域内假定的分子相互作用。
