Molecular mechanisms of IL-33 cytokine signaling

IL-1家族细胞因子在协调对感染的炎症和免疫反应方面发挥着重要作用。 然而，IL-1家族细胞因子信号转导失调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型变态反应的强效诱导剂 免疫力像其他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调节因子。 免疫细胞响应微生物入侵，在组织稳态和修复中起重要作用， 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，参与病理性纤维化反应，并与自身免疫有关。IL-33的功能是 与其同源受体ST 2结合，然后募集其第二受体IL-1RAcP。后一种受体 由其他IL-1家族细胞因子共享，最显著的是IL-1。我们最近确定了X射线晶体 小鼠IL-33/ST 2/IL-1RAcP信号传导活性三元复合物的结构。连同我们 初步的诱变，结合和功能分析，这些数据表明， IL-33和IL-1募集它们共有的二级受体IL-1RAcP的分子机制不同 很明显。这对开发能够操纵IL-10的治疗分子具有重要意义。 IL-33信号传导，以增强IL-33活化以促进有益的生理效应或抑制IL-33 信号以防止不良病理作用。我们提出的研究旨在充分证明 IL-1和IL-33信号传导的分子机制的差异，并利用这种增长机制， 工程化IL-33信号传导的新型治疗激活剂和抑制剂的知识。具体目标1： 确定IL-33细胞因子信号传导复合物形成的结构基础。在确定了水晶 为了确定鼠IL-33/ST 2/IL-1RAcP三元复合物的结构，我们现在将确定人IL-33/ST 2/IL-1RAcP三元复合物的结构。 IL-33/ST 2/IL-1RAcP三元复合物，这与我们计划的治疗设计直接相关。我们将 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）分析其构象动力学， 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 使用IL-1和IL-33。使用基于已发表的IL-1 β/IL-1RI/IL-1 β结构的结构指导方法， 1RAcP复合物和我们的新的和即将到来的IL-33/ST2/IL-1RAcP复合物的结构，我们将突变 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的结合亲和力和信号传导性质。在 具体目标3，我们将通过合理操纵IL-33信号传导机制开发新的治疗方法。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， 产生特异性和有效的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.