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.

项目总结 诺贝尔奖获奖的Toll样受体(TLRs)和白细胞介素1受体(IL-1Rs)的发现 彻底改变了我们对炎症和肿瘤发生的理解，这让我们惊讶地发现， 细胞内事件的特征仍然很差。具体地说，这两个受体家族有共同的 细胞内Toll/白介素受体(TIR)结构域与适配器TIR结构域结合以启动 信号，但还没有观察到人类寡聚体TIR复合体的结构。相反，TIR互动组 几乎完全是从单个TIR域的结构和计算得出的 建模，通常会导致数据冲突。因此，尽管自第一个结构性改革以来的近二十年 TLR1和TLR2受体的TIR结构域的特征，基础的分子机制 TIR信号的关键作用尚不清楚。我们的目标是确定TIR结构域的分子基础 作为先天免疫反应基础的相互作用，从而将启动事件连接到 具有下游事件的细胞，这些事件会导致炎症。 我们方法的新奇之处在于我们将生化、生物物理和生物学研究结合在一起 凭借我们独特的能力重组产生多个人类TIR成员，这揭示了一个令人惊讶的 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二硫键分子间交换所介导的。 半胱氨酸存在于所有TLR中，表达于细胞表面。这样的信令机制称为 只观察到PDZ结构域在细胞内形成信号复合体的情况 薄膜。然而，在其他生物和一种药物中也观察到了二硫化物介导的TIR相互作用 TLR4内相同保守的半胱氨酸的特异性阻断其活性。这类研究突出了 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，从药理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受体的抗炎活性被阻断 TLR1/TLR2通过类似的“停靠锁定”机制相互作用，为以下其中之一提供分子基础 在过去的15年里，最令人兴奋的炎症负面调节因素。 基于这些初步研究，我们假设保守的半胱氨酸调节复合体。 通过“对接锁定”机制形成TLR1、TLR2和IL-1R8。中的多功能性 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 回应。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均二聚和异二聚化的分子基础 它们的特定相互作用如何调节细胞信号。 目的2)确定IL-1R8 TIR如何阻止TLR1/TLR2 TIR相互作用以及这种相互作用如何 调节下游适配器的相互作用。