TLRs are a family of PRRs that respond to PAMPs or host-derived Danger-Associated Molecular Patterns (DAMPs) to initiate host inflammation and immune responses. TLR dimerization and recruitment of adapter molecules is critical for intracellular signaling and is mediated through intracellular Toll-Interleukin 1 Receptor Resistance (TIR) domain interactions. Human TIR domains, including reported structures of TIR1, TIR2, TIR6, TIR10, TIRAP, and MyD88, contain Cysteine (Cys) interactions or modifications that are disproportionally at, or near, reported biological TIR interfaces, or in close proximity to functionally important regions. Therefore, we hypothesized that intracellular TIR Cys regulation may have greater functional importance than previously appreciated. Expression of mutant TLR4-C747S or treatment of TLR4 reporter cells with a small molecule, Cys-binding inhibitor of TLR4, TAK-242, abrogated LPS signaling in vitro. Using TAK-242, mice were protected from lethal influenza challenge as previously reported for extracellular TLR4 antagonists. Molecular modeling and sequence analysis of the region surrounding TLR4-Cys747 indicate conservation of a WxxxE motif identified among bacterial and NAD⁺-consuming TIRs, as well as within the TIRs domains of surface TLRs 1, 2, 4, 6, and 10. Together, these data support the hypothesis that critical Cys within the TIR domain are essential for TLR4 functionality.

TLR 是一个 PRR 家族，可响应 PAMP 或宿主衍生的危险相关分子模式 (DAMP)，从而启动宿主炎症和免疫反应。 TLR 二聚化和接头分子的募集对于细胞内信号转导至关重要，并通过细胞内 Toll-白细胞介素 1 受体抗性 (TIR) 结构域相互作用介导。人类 TIR 结构域，包括已报道的 TIR1、TIR2、TIR6、TIR10、TIRAP 和 MyD88 结构，含有半胱氨酸 (Cys) 相互作用或修饰，这些相互作用或修饰在已报道的生物 TIR 界面处或附近，或在功能重要区域附近不成比例。因此，我们假设细胞内 TIR Cys 调节可能具有比之前认识到的更大的功能重要性。突变 TLR4-C747S 的表达或用小分子 TLR4 的 Cys 结合抑制剂 TAK-242 处理 TLR4 报告细胞，可在体外消除 LPS 信号传导。正如之前报道的细胞外 TLR4 拮抗剂一样，使用 TAK-242 可以保护小鼠免受致命流感攻击。 TLR4-Cys747 周围区域的分子建模和序列分析表明，在细菌和 NAD ⁺消耗 TIR 以及表面 TLR 1、2、4、6 和 10 的 TIR 结构域内发现了 WxxxE 基序的保守性。 ，这些数据支持这样的假设：TIR 域内的关键 Cys 对于 TLR4 功能至关重要。