Functional assessment of the mutational effects of human IRAK4 and MyD88 genes.
Functional assessment of the mutational effects of human IRAK4 and MyD88 genes.
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DOI：
10.1016/j.molimm.2013.11.008
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发表时间：
2014-03
影响因子：
3.6
通讯作者：
Takahiro Yamamoto;N. Tsutsumi;H. Tochio;H. Ohnishi;Kazuo Kubota;Zenichiro Kato;M. Shirakawa;N. Kond
中科院分区：
文献类型：
--
作者：
Takahiro Yamamoto;N. Tsutsumi;H. Tochio;H. Ohnishi;Kazuo Kubota;Zenichiro Kato;M. Shirakawa;N. Kond
人白细胞介素-1受体相关激酶4（IRAK 4）缺乏综合征和髓样分化因子88（MyD 88）缺乏综合征是两种具有先天性免疫缺陷的主要免疫缺陷疾病。虽然IRAK 4和MyD 88的新的遗传变异最近已被存入单核苷酸多态性（SNP）数据库，但这些变异的临床意义尚未确定。因此，重要的是建立评估每个基因变异与人类疾病的关联的方法。由于基于细胞的测定、蛋白质印迹和NF-κB报告基因测定显示R12 C和野生型IRAK 4之间的蛋白质表达和NF-κB活性没有差异，因此我们通过分析凝胶过滤和NMR滴定来检测纯化的重组IRAK 4和MyD 88蛋白的蛋白质-蛋白质相互作用。我们发现IRAK 4的变体R12 C以及R20 W，位于IRAK 4的死亡结构域，被认为是SNP，导致与MyD 88相互作用的丧失。我们的研究表明，不仅蛋白表达的缺失，而且Myddosome形成的缺陷可能导致IRAK 4和MyD 88缺乏综合征。此外，体外功能测定的组合对于确认在IRAK 4和MyD 88缺陷患者中发现的突变体的致病性是有效的。
Human interleukin-1 receptor-associated kinase 4 (IRAK4) deficiency and myeloid differentiating factor 88 (MyD88) deficiency syndromes are two primary immune-deficiency disorders with innate immune defects. Although new genetic variations ofIRAK4andMyD88have recently been deposited in the single nucleotide polymorphism (SNP) database, the clinical significance of these variants has not yet been established. Therefore, it is important to establish methods for assessing the association of each gene variation with human diseases. Because cell-based assays, western blotting and an NF-κB reporter gene assay, showed no difference in protein expression and NF-κB activity between R12C and wild-type IRAK4, we examined protein–protein interactions of purified recombinant IRAK4 and MyD88 proteins by analytical gel filtration and NMR titration. We found that the variant of IRAK4, R12C, as well as R20W, located in the death domain of IRAK4 and regarded as a SNP, caused a loss of interaction with MyD88. Our studies suggest that not only the loss of protein expression but also the defect of Myddosome formation could cause IRAK4 and MyD88 deficiency syndromes. Moreover a combination ofin vitrofunctional assays is effective for confirming the pathogenicity of mutants found in IRAK4 and MyD88-deficiency patients.