Todd Ian, Radford Paul M, Draper-Morgan Kelly-Ann, McIntosh Richard, Bainbridge Susan, Dickinson Peter, Jamhawi Lama, Sansaridis Marios, Huggins Mary L, Tighe Patrick J, Powell Richard J
Institute of Infection, Immunity and Inflammation, Division of Immunology, School of Molecular Medical Sciences, University of Nottingham, UK.
Immunology. 2004 Sep;113(1):65-79. doi: 10.1111/j.1365-2567.2004.01942.x.
Tumour necrosis factor (TNF)-receptor-associated periodic syndrome (TRAPS) is a hereditary autoinflammatory disorder involving autosomal-dominant missense mutations in TNF receptor superfamily 1A (TNFRSF1A) ectodomains. To elucidate the molecular effects of TRAPS-related mutations, we transfected HEK-293 cells to produce lines stably expressing high levels of either wild-type (WT) or single mutant recombinant forms of TNFRSF1A. Mutants with single amino acid substitutions in the first cysteine-rich domain (CRD1) were produced both as full-length receptor proteins and as truncated forms lacking the cytoplasmic signalling domain (deltasig). High-level expression of either WT or mutant full-length TNFRSF1A spontaneously induced apoptosis and interleukin-8 production, indicating that the mutations in CRD1 did not abrogate signalling. Consistent with this, WT and mutant full-length TNFRSF1A formed cytoplasmic aggregates that co-localized with ubiquitin and chaperones, and with the signal transducer TRADD, but not with the inhibitor, silencer of death domain (SODD). Furthermore, as expected, WT and mutant deltasig forms of TNFRSF1A did not induce apoptosis or interleukin-8 production. However, whereas the WT full-length TNFRSF1A was expressed both in the cytoplasm and on the cell surface, the mutant receptors showed strong cytoplasmic expression but reduced cell-surface expression. The WT and mutant deltasig forms of TNFRSF1A were all expressed at the cell surface, but a proportion of the mutant receptors were also retained in the cytoplasm and co-localized with BiP. Furthermore, the mutant forms of surface-expressed deltasig TNFRSF1A were defective in binding TNF-alpha. We conclude that TRAPS-related CRD1 mutants of TNFRSF1A possess signalling properties associated with the cytoplasmic death domain, but other behavioural features of the mutant receptors are abnormal, including intracellular trafficking and TNF binding.
肿瘤坏死因子（TNF）受体相关周期性综合征（TRAPS）是一种遗传性自身炎症性疾病，涉及TNF受体超家族1A（TNFRSF1A）胞外域的常染色体显性错义突变。为了阐明TRAPS相关突变的分子效应，我们转染了HEK - 293细胞以产生稳定表达高水平野生型（WT）或TNFRSF1A单突变重组形式的细胞系。在第一个富含半胱氨酸结构域（CRD1）中具有单氨基酸取代的突变体既作为全长受体蛋白产生，也作为缺乏细胞质信号结构域的截短形式（deltasig）产生。WT或突变全长TNFRSF1A的高水平表达自发诱导细胞凋亡和白细胞介素 - 8的产生，表明CRD1中的突变并未消除信号传导。与此一致的是，WT和突变全长TNFRSF1A形成了与泛素、伴侣蛋白以及信号转导子TRADD共定位的细胞质聚集体，但不与死亡结构域抑制剂沉默子（SODD）共定位。此外，正如预期的那样，WT和突变deltasig形式的TNFRSF1A不诱导细胞凋亡或白细胞介素 - 8的产生。然而，WT全长TNFRSF1A在细胞质和细胞表面均有表达，而突变受体则表现出强烈的细胞质表达但细胞表面表达减少。WT和突变deltasig形式的TNFRSF1A均在细胞表面表达，但一部分突变受体也保留在细胞质中并与BiP共定位。此外，表面表达的deltasig TNFRSF1A突变形式在结合TNF - α方面存在缺陷。我们得出结论，TNFRSF1A的TRAPS相关CRD1突变体具有与细胞质死亡结构域相关的信号传导特性，但突变受体的其他行为特征是异常的，包括细胞内运输和TNF结合。
