Identifying the missing link in inflammatory signaling

识别炎症信号传导中缺失的环节

• 批准号: 9807309
• 负责人: ELAN Z EISENMESSER
• 金额: $ 23.33万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-02 至 2021-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9807309
• 关键词: Address; Anti-inflammatory; Biochemical; Biological; Biophysics; Breast; Cells; Cellular Membrane; Colorectal; Complex; Computer Simulation; Conflict (Psychology); Cysteine; Data; Disulfides; Docking; Environment; Event; Family; Foundations; Generations; Goals; Heterodimerization; Homo; Homodimerization; Human; Individual; Inflammation; Inflammatory; Inflammatory Response; Innate Immune Response; Interleukin Receptor; Interleukin-1 Receptors; Ligands; Link; Liver; Malignant Neoplasms; Malignant neoplasm of lung; Mediating; Modeling; Molecular; Natural Immunity; Oranges; Organism; Orphan; Oxidation-Reduction; Oxidative Stress; Pharmaceutical Preparations; Pharmacology; Prostate; Receptor Signaling; Recombinants; Role; Signal Transduction; Stomach; Structure; Surface; TLR1 gene; TLR2 gene; TLR4 gene; Therapeutic; Therapeutic Intervention; Toll-like receptors; Yersinia pestis; base; intimate behavior; member; novel; overexpression; receptor; receptor-mediated signaling; tumorigenesis

PROJECT SUMMARY The Nobel winning discoveries of Toll-like Receptors (TLRs) and interleukin-1 receptors (IL-1Rs) have revolutionized our understanding of inflammation and oncogenesis, which makes it surprising that the initiating intracellular events still remain poorly characterized. Specifically, both receptor families share common intracellular Toll/Interleukin-Receptor (TIR) domains that engage adaptor TIR domains in order to initiate signaling, yet no human oligomeric TIR complex has been structurally observed. Instead, the TIR interactome has almost exclusively been extrapolated from structures of individual TIR domains and computational modeling, often resulting in conflicting data. Thus, despite the nearly two decades since the first structural characterization of the TIR domains of TLR1 and TLR2 receptors, the molecular mechanisms that underlie the critical roles of TIR signaling remains unknown. Our goals are to determine the molecular basis of TIR domain interactions that underlie the innate immune response, thereby bridging the initiating events on the outside of the cell with downstream events that drive inflammation. The novelty in our approach is our combination of biochemical, biophysical, and biological studies along with our unique ability to recombinantly produce multiple human TIR members, which has revealed a surprising underlying molecular mechanism of TLR interactions. Namely, we have discovered that TLR1/TLR2 homodimer and heterodimer formation are mediated by an intermolecular disulfide exchange of a conserved cysteine found in all TLRs expressed on the cellular surface. Such a signaling mechanism, referred to as “dock-and-lock”, has only been observed for PDZ domains that also form signaling complexes at the cellular membrane. However, disulfide mediated TIR interactions have been observed in other organisms and a drug specific for the same conserved cysteine within TLR4 blocks its activity. Such studies highlight the importance of determining the molecular mechanism of TLR interactions that would also provide a basis for pharmacologically blocking their interactions through targeting of this conserved cysteine. Interestingly, our preliminary studies also suggest that the anti-inflammatory activity of the orphaned IL-1R8 receptor blocks TLR1/TLR2 interactions through a similar “dock-and-lock” mechanism, providing the molecular basis for one of the most exciting negative regulators of inflammation within the last 15 years. Based on these preliminary studies, we hypothesize that a conserved cysteine mediates complex formation of TLR1, TLR2, and IL-1R8 through a “dock-and-lock” mechanism. The versatility in homo/heterodimerization results in varied downstream TIR interactions that fine-tune the cellular inflammatory response. We will address this hypothesis through the following Specific Aims: Aim 1) Determine the molecular basis of TLR1/TLR2 TIR homodimerization and heterodimerization and how their specific interactions regulate cellular signaling. Aim 2) Determine how IL-1R8 TIR blocks TLR1/TLR2 TIR interactions and how such interactions modulate downstream adaptor interactions.

项目概要 Toll 样受体 (TLR) 和白细胞介素 1 受体 (IL-1R) 的诺贝尔奖发现 彻底改变了我们对炎症和肿瘤发生的理解，这令人惊讶 细胞内事件的特征仍然很差。具体来说，两个受体家族都有共同点 细胞内 Toll/白细胞介素受体 (TIR) 结构域与适配器 TIR 结构域结合以启动 信号传导，但尚未观察到人类寡聚 TIR 复合物的结构。相反，TIR 相互作用组 几乎完全是从各个 TIR 域的结构和计算 建模，通常会导致数据冲突。因此，尽管自第一次结构性 TLR1 和 TLR2 受体 TIR 结构域的表征，以及 TLR1 和 TLR2 受体的分子机制 TIR 信号传导的关键作用仍然未知。我们的目标是确定 TIR 结构域的分子基础 先天免疫反应基础上的相互作用，从而桥接外部的起始事件 具有驱动炎症的下游事件的细胞。 我们方法的新颖之处在于我们将生物化学、生物物理和生物学研究相结合 凭借我们重组产生多个人类 TIR 成员的独特能力，这揭示了令人惊讶的 TLR 相互作用的潜在分子机制。也就是说，我们发现TLR1/TLR2 同二聚体和异二聚体的形成是由保守的分子间二硫键交换介导的 半胱氨酸存在于细胞表面表达的所有 TLR 中。这种信号机制，称为 “对接和锁定”，仅在 PDZ 结构域中观察到，该结构域也在细胞中形成信号复合物 膜。然而，二硫键介导的 TIR 相互作用已在其他生物体和药物中观察到 对 TLR4 内相同保守的半胱氨酸具有特异性，可阻断其活性。此类研究强调了重要性 确定 TLR 相互作用的分子机制，这也将为 通过靶向这种保守的半胱氨酸在药理学上阻断它们的相互作用。有趣的是，我们的 初步研究还表明，孤儿 IL-1R8 受体的抗炎活性可阻断 TLR1/TLR2 通过类似的“对接锁定”机制相互作用，为其中之一提供了分子基础 过去 15 年来最令人兴奋的炎症负调节因子。 基于这些初步研究，我们假设保守的半胱氨酸介导复杂的 通过“对接锁定”机制形成 TLR1、TLR2 和 IL-1R8。多功能性 同源/异源二聚化导致不同的下游 TIR 相互作用，从而微调细胞炎症 回复。我们将通过以下具体目标来解决这一假设： 目的 1) 确定 TLR1/TLR2 TIR 同二聚和异二聚的分子基础 它们的特定相互作用如何调节细胞信号传导。 目标 2) 确定 IL-1R8 TIR 如何阻断 TLR1/TLR2 TIR 相互作用以及这种相互作用如何发生 调节下游适配器的相互作用。