Extracellular trafficking of tumor necrosis factor receptor superfamily (TNFRSF) is tightly regulated, disruption of which triggers various autoinflammatory disorders, including TNF receptor‐associated periodic syndrome (TRAPS). Here, we provide thus far unraveled molecular basis of noncysteine mutations in TNFR1 ectodomain where loss of an aromatic moiety in cysteine‐rich domain (CRD) 2 results in TRAPS disease‐associated phenotype. Our study characterized that a missense mutation on phenylalanine residue located in CRD2 (TNFR1F60V) causes a delay in TNFR1 transport to cell membrane, leading to sustained receptor responsiveness and downstream NF‐κB activation, characteristic of clinical manifestation of a prolonged fever. By creating and characterizing identical mutations on structurally conserved ectodomains of osteoprotegerin (OPG) and decoy receptor 3, other two secreted forms of TNFRSF, we further identified that a conserved aromatic residue at the A1 submodule of CRD2 (A1CRD2) confers structural integrity of ectodomain where aromatic sidechain deletion increases thermal instability, interfering with efficient posttranslational modification and subsequent receptor secretion. Interestingly, our functional analyses indicated that this particular noncysteine mutation is not associated with either protein misfolding or loss of function. Finally, by using a synthetic agonist, we demonstrated gain‐of‐function of the trafficking defect, suggesting the possibility of rescuing affected pathology in related disorders. Given the structural and topological similarities present in the ectodomains of TNFRSF members, our findings provide mechanistic insights of defects in subcellular trafficking of TNF receptors, reported in various TNFRSF‐associated diseases.

中文翻译：

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胞外域中的保守芳香族部分是 TNFR 超家族结构完整性和蛋白质运输的关键决定因素

肿瘤坏死因子受体超家族 (TNFRSF) 的细胞外运输受到严格调控，其破坏会引发各种自身炎症性疾病，包括 TNF 受体相关周期性综合征 (TRAPS)。在这里，我们提供了迄今为止尚未阐明的 TNFR1 胞外域非半胱氨酸突变的分子基础，其中富含半胱氨酸结构域 (CRD) 2 中芳香族部分的丢失导致 TRAPS 疾病相关表型。我们的研究表明，位于 CRD2 (TNFR1F60V) 的苯丙氨酸残基上的错义突变导致 TNFR1 转运至细胞膜的延迟，导致持续的受体反应和下游 NF-κB 激活，这是长期发烧的临床表现特征。通过在骨保护素 (OPG) 和诱饵受体 3（TNFRSF 的另外两种分泌形式）的结构保守的胞外域上创建并表征相同的突变，我们进一步确定了 CRD2 (A1CRD2) 的 A1 子模块上的保守芳香族残基赋予了胞外域的结构完整性，其中芳香族侧链缺失增加了热不稳定性，干扰有效的翻译后修饰和随后的受体分泌。有趣的是，我们的功能分析表明，这种特殊的非半胱氨酸突变与蛋白质错误折叠或功能丧失无关。最后，通过使用合成激动剂，我们证明了运输缺陷的功能获得，这表明有可能挽救相关疾病中受影响的病理。鉴于 TNFRSF 成员胞外域中存在的结构和拓扑相似性，我们的研究结果提供了在各种 TNFRSF 相关疾病中报道的 TNF 受体亚细胞运输缺陷的机制见解。