INSERM U1163 Paris-Descartes University, Sorbonne Paris Cité, IMAGINE Institute, Necker Hospital Enfants-Malades, Paris, France.
Laboratory of Signaling and Pathogenesis, CNRS UMR 3691, Pasteur Institute, Paris, France.
J Allergy Clin Immunol. 2017 Dec;140(6):1671-1682.e2. doi: 10.1016/j.jaci.2016.11.056. Epub 2017 Feb 27.
Incontinentia pigmenti (IP; MIM308300) is a severe, male-lethal, X-linked, dominant genodermatosis resulting from loss-of-function mutations in the IKBKG gene encoding nuclear factor κB (NF-κB) essential modulator (NEMO; the regulatory subunit of the IκB kinase [IKK] complex). In 80% of cases of IP, the deletion of exons 4 to 10 leads to the absence of NEMO and total inhibition of NF-κB signaling. Here we describe a new IKBKG mutation responsible for IP resulting in an inactive truncated form of NEMO.
We sought to identify the mechanism or mechanisms by which the truncated NEMO protein inhibits the NF-κB signaling pathway.
We sequenced the IKBKG gene in patients with IP and performed complementation and transactivation assays in NEMO-deficient cells. We also used immunoprecipitation assays, immunoblotting, and an in situ proximity ligation assay to characterize the truncated NEMO protein interactions with IKK-α, IKK-β, TNF receptor-associated factor 6, TNF receptor-associated factor 2, receptor-interacting protein 1, Hemo-oxidized iron regulatory protein 2 ligase 1 (HOIL-1), HOIL-1-interacting protein, and SHANK-associated RH domain-interacting protein. Lastly, we assessed NEMO linear ubiquitination using immunoblotting and investigated the formation of NEMO-containing structures (using immunostaining and confocal microscopy) after cell stimulation with IL-1β.
We identified a novel splice mutation in IKBKG (c.518+2T>G, resulting in an in-frame deletion: p.DelQ134_R256). The mutant NEMO lacked part of the CC1 coiled-coil and HLX2 helical domain. The p.DelQ134_R256 mutation caused inhibition of NF-κB signaling, although the truncated NEMO protein interacted with proteins involved in activation of NF-κB signaling. The IL-1β-induced formation of NEMO-containing structures was impaired in fibroblasts from patients with IP carrying the truncated NEMO form (as also observed in HOIL-1 cells). The truncated NEMO interaction with SHANK-associated RH domain-interacting protein was impaired in a male fetus with IP, leading to defective linear ubiquitination.
We identified a hitherto unreported disease mechanism (defective linear ubiquitination) in patients with IP.
色素失禁症（IP；MIM308300）是一种严重的、男性致死的、X 连锁显性遗传性皮肤病，由 NF-κB（核因子 κB）必需调节剂（NEMO；IKK 复合物的调节亚基）基因 IKBKG 编码的功能丧失突变引起。在 80%的 IP 病例中，外显子 4 到 10 的缺失导致 NEMO 缺失和 NF-κB 信号的完全抑制。在这里，我们描述了一种导致 IP 的新的 IKBKG 突变，导致 NEMO 的无活性截断形式。
我们试图确定截断的 NEMO 蛋白抑制 NF-κB 信号通路的机制或机制。
我们对 IP 患者的 IKBKG 基因进行了测序，并在 NEMO 缺陷细胞中进行了互补和转激活测定。我们还使用免疫沉淀测定、免疫印迹和原位邻近连接测定来描述截断的 NEMO 蛋白与 IKK-α、IKK-β、TNF 受体相关因子 6、TNF 受体相关因子 2、受体相互作用蛋白 1、血红素氧化酶调节蛋白 2 连接酶 1（HOIL-1）、HOIL-1 相互作用蛋白和 SHANK 相关 RH 结构域相互作用蛋白的相互作用。最后，我们使用免疫印迹评估了 NEMO 的线性泛素化，并研究了在细胞受到 IL-1β刺激后形成的包含 NEMO 的结构（使用免疫染色和共聚焦显微镜）。
我们在 IKBKG 中发现了一种新的剪接突变（c.518+2T>G，导致框内缺失：p.DelQ134_R256）。突变的 NEMO 缺失了部分 CC1 卷曲螺旋和 HLX2 螺旋结构域。p.DelQ134_R256 突变导致 NF-κB 信号转导受到抑制，尽管截断的 NEMO 蛋白与参与 NF-κB 信号转导激活的蛋白相互作用。在携带截断 NEMO 形式的 IP 患者的成纤维细胞中，IL-1β 诱导的包含 NEMO 的结构的形成受到损害（在 HOIL-1 细胞中也观察到）。在一名患有 IP 的男性胎儿中，截断的 NEMO 与 SHANK 相关 RH 结构域相互作用蛋白的相互作用受损，导致线性泛素化缺陷。
我们在 IP 患者中发现了一种以前未报道的疾病机制（缺陷的线性泛素化）。
