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.

项目摘要 诺贝尔奖的诺贝尔奖励受体（TLR）和白介素-1受体（IL-1R）的发现具有 彻底改变了我们对炎症和肿瘤发生的理解，这使得启动使我们感到惊讶 细胞内事件的特征仍然很差。具体而言，两个受体家族共同 细胞内收费/白介素 - 受体（TIR）域，这些域吸引了适配器TIR域以启动 信号传导，但没有在结构上观察到人类的低聚TIR复合物。相反，tir互动 几乎完全从单个TIR域和计算的结构中推断出 建模，通常会导致数据冲突。这是自第一个结构以来近二十年来绝望的 TLR1和TLR2受体的TIR结构域的表征，这是分子机制 TIR信号的关键作用仍然未知。我们的目标是确定TIR域的分子基础 具有先天免疫反应的基础的互动，从而弥合了在外面的启动事件 具有驱动炎症的下游事件的细胞。 我们方法的新颖性是我们沿着生化，生物物理和生物学研究的结合 我们具有重组生产多个人类TIR成员的独特能力，这表明了一个惊喜 TLR相互作用的基本分子机制。即，我们发现TLR1/TLR2 同二聚体和异二聚体形成是通过保守的分子间二硫化物交换来介导的 在细胞表面表达的所有TLR中发现的半胱氨酸。这样的信号机制，称为 仅在PDZ域中观察到“ Dock and Lock”，该域也形成了在细胞处的信号复合物 膜。但是，在其他生物体和药物中已经观察到了二硫键介导的TIR相互作用 特定于TLR4中同一配置的半胱氨酸的特定于其活性。这样的研究强调了重要性 确定TLR相互作用的分子机制，这也将为 药理通过靶向这种保守的半胱氨酸来阻止其相互作用。有趣的是，我们的 初步研究还表明，孤立的IL-1R8受体块的抗炎活性 TLR1/TLR2通过类似的“ dock and-lock”机制相互作用，为其中一个提供了分子基础 过去15年内，最令人兴奋的炎症负面调节因子。 基于这些初步研究，我们假设构成半胱氨酸会介导复合物 TLR1，TLR2和IL-1R8的形成通过“ dock and-lock”机制。多功能性 同型/异二聚化导致不同的下游TIR相互作用，以微调细胞炎症 回复。我们将通过以下特定目的解决这一假设： 目标1）确定TLR1/TLR2 TIR均二聚化和异二聚化和异构化的分子基础 他们的特定相互作用如何调节细胞信号。 目标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 调节下游适配器相互作用。