Molecular mechanisms of IL-33 cytokine signaling

IL-1家族细胞因子在对感染的炎症和免疫反应中起重要作用。 然而，调节失调的IL-1家族细胞因子信号是许多慢性炎症性疾病的关键因素 疾病和自身免疫性疾病。IL-33是IL-1家族成员，是2型过敏症的有效诱因 豁免权。像其他IL-1家族细胞因子一样，它对人类健康有积极影响--它激活了广泛的 免疫细胞对微生物入侵的反应，在组织动态平衡和修复中发挥重要作用，以及 逆转阿尔茨海默病小鼠模型的症状；但也会产生负面影响-它促进 过敏性哮喘，参与病理纤维化反应，并与自身免疫有关。IL-33的功能由 与其同源受体ST2结合，然后招募其第二受体IL-1RAcP。后一种受体 被其他IL-1家族细胞因子所共享，最显著的是IL-1。我们最近确定了X射线晶体 小鼠IL-33/ST2/IL-1RAcP信号活性三元复合体的结构与我们的 初步的诱变、结合和功能分析，这些数据表明假设 IL-33和IL-1募集其共有的次级受体IL-1RAcP的分子机制不同 很明显。这对开发能够操纵IL-1的治疗分子具有重要意义。 33信号，增强IL-33的激活以促进有益的生理效应或抑制IL-33 发出信号以防止不利的病理影响。我们建议的研究旨在充分证明 IL-1和IL-33信号转导的分子机制差异及其作用机制 设计新型IL-33信号治疗激活剂和抑制剂的知识。在具体目标1中，我们将 确定IL-33细胞因子信号复合体形成的结构基础。在确定了晶体之后 小鼠IL-33/ST2/IL-1RAcP三元复合物的结构，现在我们将确定人的结构 IL-33/ST2/IL-1RAcP三元复合体，这与我们计划的治疗设计直接相关。我们会 还用小角X射线散射(SAXS)评价了这些络合物的溶液结构，并对 氢/氢交换质谱仪(HDX-MS)分析它们的构象动力学 分子动力学(MD)模拟。在特定的目标2中，我们将定义共享受体的分子基础 IL-1和IL-33的使用情况。使用基于已发表的IL-1/IL-1RI/IL-1结构的结构导向方法 1RAcP复合体和IL-33/ST2/IL-1RAcP复合体的新结构，我们将突变 复合细胞因子/同源受体和辅助蛋白形成的界面上的残基 并测量它们与野生型蛋白的结合亲和力和信号特性。在……里面 具体目标3，我们将通过合理操作IL-33信号机制来开发新的治疗方法。我们 将使用各种定向进化、基于结构的蛋白质设计和抗体工程方法来 产生特异性和强效的IL-33信号激活剂和抑制物。

IL-1 family cytokines are instrumental in orchestrating inflammatory and immune responses to infection. However, dysregulated IL-1 family cytokine signaling is a key contributor to numerous chronic inflammatory diseases and autoimmune disorders. IL-33, an IL-1 family member, is a potent inducer of allergic type 2 immunity. Like other IL-1 family cytokines, it positively impacts human health – it activates a wide range of immune cells in response to microbial invasion, plays important roles in tissue homeostasis and repair, and reverses symptoms in mouse models of Alzheimer’s disease; but also drives negative impacts – it promotes allergic asthma, participates in pathological fibrotic reactions, and is linked to autoimmunity. IL-33 functions by binding to its cognate receptor, ST2, and then recruiting its secondary receptor, IL-1RAcP. The latter receptor is shared by other IL-1 family cytokines, most notably IL-1. We have recently determined the X-ray crystal structure of the murine IL-33/ST2/IL-1RAcP signaling-competent ternary complex. Together with our preliminary mutagenesis, binding and functional analyses, these data suggest the hypothesis that the molecular mechanisms by which IL-33 and IL-1 recruit their shared secondary receptor, IL-1RAcP, differ markedly. This has important implications for the development of therapeutic molecules that can manipulate IL- 33 signaling, either to augment IL-33 activation to promote beneficial physiological effects or to inhibit IL-33 signaling to prevent adverse pathological effects. Our proposed studies are designed to fully demonstrate the differences in molecular mechanisms of IL-1 and IL-33 signaling and to leverage this growing mechanistic knowledge to engineer novel therapeutic activators and inhibitors of IL-33 signaling. In Specific Aim 1, we will determine the structural basis of IL-33 cytokine signaling complex formation. Having determined the crystal structure of the murine IL-33/ST2/IL-1RAcP ternary complex, we will now determine the structure of the human IL-33/ST2/IL-1RAcP ternary complex, which is directly relevant to our planned therapeutic designs. We will also evaluate the solution structures of these complexes by small-angle X-ray scattering (SAXS) and assess their conformational dynamics by hydrogen/deuterium exchange-mass spectrometry (HDX-MS) analysis and molecular dynamics (MD) simulations. In Specific Aim 2, we will define the molecular basis of shared receptor usage by IL-1 and IL-33. Using a structure-guided approach based on published structures of IL-1/IL-1RI/IL- 1RAcP complexes and our new and forthcoming structures of IL-33/ST2/IL-1RAcP complexes, we will mutate residues within the interfaces formed by the composite cytokine/cognate receptor and accessory protein surfaces, and measure their binding affinities and signaling properties relative to the wild type proteins. In Specific Aim 3, we will develop novel therapeutics by rationally manipulating IL-33 signaling mechanisms. We will use a variety of directed evolution, structure-based protein design, and antibody engineering methods to produce specific and potent activators and inhibitors of IL-33 signaling.