Quantitative single‐molecule imaging of TNFR1 reveals zafirlukast as antagonist of TNFR1 clustering and TNFα‐induced NF‐ĸB signaling
Quantitative single‐molecule imaging of TNFR1 reveals zafirlukast as antagonist of TNFR1 clustering and TNFα‐induced NF‐ĸB signaling
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TNFR1 的定量单分子成像揭示扎鲁司特作为 TNFR1 簇和 TNFα 诱导的 NF-B 信号传导的拮抗剂
DOI：
10.1002/jlb.2ab0420-572rr
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发表时间：
2020
影响因子：
5.5
通讯作者：
van Wijk SJL
中科院分区：
文献类型：
--
作者：
Weinelt N;Karathanasis C;Smith S;Medler J;Malkusch S;Fulda S;Wajant H;Heilemann M;van Wijk SJL
TNFR 1是NF-FIB介导的促炎细胞存活应答和程序性细胞死亡（PCD）的关键调节剂。TNFα和TNFR 1控制的NF-κ B信号的失调是主要疾病的基础，如癌症、炎症和自身免疫性疾病。因此，尽管TNFα拮抗剂被常规使用，但也可能影响TNFR 2介导的过程，因此直接拮抗TNFR 1的替代方法是有益的。在这里，我们应用定量单分子定位显微镜（SMLM）的TNFR 1在生理细胞环境中验证和表征TNFR 1抑制物质，最近描述的TNFR 1拮抗剂扎鲁司特为例。用扎鲁司特处理TNFR 1-mEos 2重建的TNFR 1/2敲除小鼠胚胎成纤维细胞（MEF），可抑制配体非依赖性前配体组装结构域（PLAD）介导的TNFR 1二聚化以及TNFα诱导的TNFR 1寡聚化。此外，扎鲁司特介导的TNFR 1聚集抑制伴随着重组TNFR 1-mEos 2 MEFs和人宫颈癌细胞中急性和长期NF-KB信号转导的失调。这些发现揭示了PLAD介导的、配体非依赖性TNFR 1二聚化对于NF-κ B活化的必要性，突出了PLAD作为TNFα诱导的TNFR 1寡聚化的中心调节剂，并证明TNFR 1-mEos 2 MEFs可用于在生理条件下采用单分子定量和功能性NF-κ B测定来研究TNFR 1拮抗化合物。
TNFR1 is a crucial regulator of NF-ĸB-mediated proinflammatory cell survival responses and programmed cell death (PCD). Deregulation of TNFα- and TNFR1-controlled NF-ĸB signaling underlies major diseases, like cancer, inflammation, and autoimmune diseases. Therefore, although being routinely used, antagonists of TNFα might also affect TNFR2-mediated processes, so that alternative approaches to directly antagonize TNFR1 are beneficial. Here, we apply quantitative single-molecule localization microscopy (SMLM) of TNFR1 in physiologic cellular settings to validate and characterize TNFR1 inhibitory substances, exemplified by the recently described TNFR1 antagonist zafirlukast. Treatment of TNFR1-mEos2 reconstituted TNFR1/2 knockout mouse embryonic fibroblasts (MEFs) with zafirlukast inhibited both ligand-independent preligand assembly domain (PLAD)-mediated TNFR1 dimerization as well as TNFα-induced TNFR1 oligomerization. In addition, zafirlukast-mediated inhibition of TNFR1 clustering was accompanied by deregulation of acute and prolonged NF-ĸB signaling in reconstituted TNFR1-mEos2 MEFs and human cervical carcinoma cells. These findings reveal the necessity of PLAD-mediated, ligand-independent TNFR1 dimerization for NF-ĸB activation, highlight the PLAD as central regulator of TNFα-induced TNFR1 oligomerization, and demonstrate that TNFR1-mEos2 MEFs can be used to investigate TNFR1-antagonizing compounds employing single-molecule quantification and functional NF-ĸB assays at physiologic conditions.
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