Department of Pediatrics, Graduate School of Medicine, Gifu University, 1-1 Yanagido, Gifu 501-1194, Japan.
Department of Molecular Engineering, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan.
Mol Immunol. 2014 Mar;58(1):66-76. doi: 10.1016/j.molimm.2013.11.008. Epub 2013 Dec 5.
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 of IRAK4 and MyD88 have 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 of in vitro functional assays is effective for confirming the pathogenicity of mutants found in IRAK4 and MyD88-deficiency patients.
人白细胞介素-1 受体相关激酶 4 (IRAK4) 缺陷和髓样分化因子 88 (MyD88) 缺陷综合征是两种具有固有免疫缺陷的原发性免疫缺陷病。尽管 IRAK4 和 MyD88 的新遗传变异最近已被提交到单核苷酸多态性 (SNP) 数据库，但这些变异的临床意义尚未确定。因此，建立评估每个基因变异与人类疾病关联的方法非常重要。由于细胞测定、western blot 和 NF-κB 报告基因测定显示 R12C 和野生型 IRAK4 之间的蛋白表达和 NF-κB 活性没有差异，因此我们通过分析凝胶过滤和 NMR 滴定来检查纯化的重组 IRAK4 和 MyD88 蛋白的蛋白-蛋白相互作用。我们发现 IRAK4 的变体 R12C，以及位于 IRAK4 的死亡域中的 R20W，被认为是 SNP，导致与 MyD88 的相互作用丧失。我们的研究表明，不仅是蛋白表达的丧失，而且 Myddosome 形成的缺陷也可能导致 IRAK4 和 MyD88 缺陷综合征。此外，体外功能测定的组合可有效确认在 IRAK4 和 MyD88 缺陷患者中发现的突变体的致病性。
