Functional studies for the TRAF6 mutation associated with hypohidrotic ectodermal dysplasia
Functional studies for the TRAF6 mutation associated with hypohidrotic ectodermal dysplasia
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DOI：
10.1111/bjd.12018
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发表时间：
2013-03-01
影响因子：
10.3
通讯作者：
Shimomura, Y.
中科院分区：
文献类型：
--
作者：
Fujikawa, H.;Farooq, M.;Shimomura, Y.
背景 少汗性外胚层发育不良（HED）是一种罕见疾病，其特征为少毛症、少汗症和缺牙症。最近在一名 HED 患者中发现了肿瘤坏死因子受体相关因子 6 基因 (TRAF6) 的从头杂合突变，但该突变导致的功能后果仍未知。 目的 确定 TRAF6 突变导致 HED 的机制。 方法 我们进行了免疫共沉淀 (co-IP) 研究，以确定该突变是否会影响 TRAF6 与转化生长因子 β 激活激酶 1 (TAK1) 的相互作用， TAK1 结合蛋白 2 (TAB 2) 和外胚层增生素 A 受体相关死亡结构域蛋白 (EDARADD)。然后，我们进行了 co-IP 和谷胱甘肽 S-转移酶下拉测定，以确定 EDARADD 中的 TRAF6 结合序列。此外，我们分析了突变型TRAF6蛋白对野生型TRAF6和EDARADD之间的亲和力以及EDARADD介导的核因子（NF）-κB激活的影响。结果突变型TRAF6蛋白能够以与野生型TRAF6类似的方式与TAK1和TAB 2形成复合物。然而，突变型TRAF6蛋白完全失去了对EDARADD的亲和力，而野生型TRAF6则与EDARADD的N端结构域结合。此外，突变体TRAF6抑制野生型TRAF6和EDARADD之间的相互作用，并且还可能降低EDARADD介导的NF-κB活性。结论我们得出结论，突变体TRAF6蛋白对野生型TRAF6蛋白表现出显性负作用，预计这会影响外胚层衍生器官发育过程中EDARADD介导的NF-κB激活，并导致HED表型。
Background Hypohidrotic ectodermal dysplasia (HED) is a rare condition characterized by hypotrichosis, hypohidrosis and hypodontia. A de novo heterozygous mutation in the tumour necrosis factor receptor-associated factor 6 gene (TRAF6) was recently identified in a patient with HED, while functional consequences resulting from the mutation remained unknown.Objectives To determine the mechanism by which the TRAF6 mutation results in HED.Methods We performed coimmunoprecipitation (co-IP) studies to determine whether the mutation would affect the interaction of TRAF6 with transforming growth factor beta-activated kinase 1 (TAK1), TAK1-binding protein 2 (TAB 2) and ectodysplasin-A receptor-associated death domain protein (EDARADD). We then performed co-IP and glutathione S-transferase-pulldown assays to determine the TRAF6 binding sequences in EDARADD. In addition, we analysed the effect of the mutant TRAF6 protein on the affinity between wild-type TRAF6 and EDARADD, as well as on EDARADD-mediated nuclear factor (NF)-kappa B activation.Results The mutant TRAF6 protein was capable of forming a complex with TAK1 and TAB 2 in a similar way to wild-type TRAF6. However, the mutant TRAF6 protein completely lost the affinity to EDARADD, while the wild-type TRAF6 bound to the N-terminal domain of EDARADD. Furthermore, the mutant TRAF6 inhibited the interaction between the wild-type TRAF6 and EDARADD, and also potentially reduced the EDARADD-mediated NF-kappa B activity.Conclusions We conclude that the mutant TRAF6 protein shows a dominant negative effect against the wild-type TRAF6 protein, which is predicted to affect the EDARADD-mediated activation of NF-kappa B during the development of ectoderm-derived organs, and to lead to the HED phenotype.