Molecular mechanisms of IL-33 cytokine signaling

IL-1 家族细胞因子有助于协调针对感染的炎症和免疫反应。 然而，IL-1 家族细胞因子信号传导失调是许多慢性炎症的关键因素。 疾病和自身免疫性疾病。 IL-33 是 IL-1 家族成员，是 2 型过敏的有效诱导剂 免疫力。与其他 IL-1 家族细胞因子一样，它对人类健康产生积极影响 – 它激活多种细胞因子 免疫细胞响应微生物入侵，在组织稳态和修复中发挥重要作用， 逆转阿尔茨海默病小鼠模型的症状；但也带来了负面影响——它促进了 过敏性哮喘，参与病理性纤维化反应，并与自身免疫相关。 IL-33 的功能是 与其同源受体 ST2 结合，然后招募其次级受体 IL-1RAcP。后一种受体 与其他 IL-1 家族细胞因子共享，尤其是 IL-1。我们最近确定了X射线晶体 鼠 IL-33/ST2/IL-1RAcP 信号传导三元复合物的结构。与我们一起 初步诱变、结合和功能分析，这些数据提出了这样的假设： IL-33 和 IL-1 募集其共享的次级受体 IL-1RAcP 的分子机制不同 明显。这对于开发能够操纵 IL-的治疗分子具有重要意义。 33 信号传导，增强 IL-33 激活以促进有益的生理效应或抑制 IL-33 信号传导以防止不良病理效应。我们提出的研究旨在充分证明 IL-1 和 IL-33 信号传导分子机制的差异，并利用这种不断发展的机制 设计新型治疗性 IL-33 信号传导激活剂和抑制剂的知识。在具体目标 1 中，我们将 确定 IL-33 细胞因子信号传导复合物形成的结构基础。确定晶体后 鼠IL-33/ST2/IL-1RAcP三元复合物的结构，我们现在将确定人类IL-33/ST2/IL-1RAcP三元复合物的结构 IL-33/ST2/IL-1RAcP 三元复合物，与我们计划的治疗设计直接相关。我们会 还通过小角 X 射线散射 (SAXS) 评估这些配合物的溶液结构并评估 通过氢/氘交换质谱 (HDX-MS) 分析它们的构象动力学 分子动力学（MD）模拟。在具体目标 2 中，我们将定义共享受体的分子基础 IL-1 和 IL-33 的使用。使用基于已发表的 IL-1/IL-1RI/IL- 结构的结构引导方法 1RAcP 复合物以及我们即将推出的新的 IL-33/ST2/IL-1RAcP 复合物结构，我们将进行突变 由复合细胞因子/同源受体和辅助蛋白形成的界面内的残基 表面，并测量它们相对于野生型蛋白质的结合亲和力和信号传导特性。在 具体目标 3，我们将通过合理操纵 IL-33 信号传导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 产生特定且有效的 IL-33 信号传导激活剂和抑制剂。

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.