Molecular Recognition Studies of the Interleukin-7 Pathway

描述(申请人提供)：白介素7(IL-7)是一种重要的多营养细胞因子，调节多种免疫事件。正常的IL-7信号反应触发T和B细胞的发育、增殖和动态平衡，以及细胞外基质的重塑。IL-7通过与其自身的细胞表面受体IL-7Rα和常见的伽马链受体相互作用，通过细胞因子诱导受体异源二聚化机制激活其信号级联反应。IL-7途径的过低和过度刺激与一种严重的联合免疫缺陷、自身免疫疾病、冠状动脉疾病和几种癌症的发病机制有关。对IL-7诱导的受体异构化的结构、能量和细胞生物学研究需要仔细分析两个不同的事件：IL-7与IL-7Rα的关联以及IL-7：IL-7Rα复合体与常见的伽马链受体的关联。这项研究计划将重点放在IL-7与其α受体的结合作用的起始步骤。结果表明，IL-7Rα的糖基化赋予了与IL-7最佳的结合亲和力。IL-7Rα糖基化的重要性似乎是常见的伽马链受体家族的一种新的结合识别机制。野生型IL-7：IL-7Rα相互作用的热力学和动力学性质将使用各种生物物理方法来确定，包括等温滴定、量热法和表面等离子体共振。IL-7和IL-7Rα的功能表位将使用定点扫描突变、生物物理方法和细胞增殖分析来确定。我们将使用X射线结晶学或核磁共振光谱来确定游离IL-7、游离未糖化和糖基化的IL-7Rα以及与未糖化和糖基化的IL-7Rα结合的IL-7的复合体的三维结构。我们对IL-7-受体相互作用结合决定因素的理解将被用于利用合理的蛋白质设计和噬菌体展示技术设计新型多肽和蛋白质拮抗剂。这项研究的长期目标包括开发一个结构能量模型来解释正常和异常的IL-7信号机制，以及产生新的治疗性多肽/蛋白质来治疗与IL-7信号受损相关的许多缺陷和疾病。 公共卫生相关声明：这项研究建议将重点放在结构、能量和细胞生物学研究上，研究人白介素7及其受体白介素7受体α之间的相互作用。我们对IL-7-受体相互作用结合决定因素的理解将被用于利用合理的蛋白质设计和噬菌体展示技术设计新型多肽和蛋白质拮抗剂。这项研究的长期目标包括开发一个结构能量模型来解释正常和异常的IL-7信号机制，以及产生新的治疗性多肽/蛋白质来治疗与人IL-7信号受损相关的许多缺陷和疾病。

DESCRIPTION (provided by applicant): Interleukin-7 (IL-7) is an essential pleiotrophic cytokine regulating numerous immunological events. Normal IL-7 signaling responses trigger the development, proliferation, and homeostasis of T and B cells as well as remodeling of the extracellular matrix. IL-7 activates its signaling cascade by interacting with its own cell surface receptor, IL-7R alpha, and the common gamma chain receptor through a cytokine-induced receptor heterodimerization mechanism. Under- and over-stimulation of the IL-7 pathway has been implicated in the pathogenesis of a form of severe combined immunodeficiency, autoimmune conditions, coronary artery disease, and several cancers. Structural, energetic, and cell biological studies of IL-7-induced receptor heterodimerization require a careful analysis of two distinct events: the association of IL-7 to IL-7R alpha and of IL-7:IL-7R alpha complex to the common gamma chain receptor. This research proposal will focus on the initiation step, the binding interaction of IL-7 with its alpha receptor. Results show that glycosylation of the IL- 7R alpha confers optimal binding affinity to IL-7. The importance of glycosylation of the IL-7R alpha appears to be a novel binding recognition mechanism for the common gamma chain receptor family. The thermodynamic and kinetic properties of the wild-type IL-7:IL-7R alpha interaction will be ascertained using a variety of biophysical methods including isothermal titration calorimetry and surface plasmon resonance. The functional epitopes on both IL-7 and IL-7R alpha will be determined using site-directed scanning mutagenesis, biophysical methods, and cell proliferation assays. Three-dimensional structures of free IL-7, free unglycosylated and glycosylated IL-7R alpha, and complexes of IL-7 bound to unglycosylated and glycosylated IL-7R alpha will be determined using x-ray crystallography or NMR spectroscopy. Our understanding of the binding determinants for the IL-7-receptor interaction will be used to engineer novel peptide and protein antagonists using rational protein design and phage display techniques. The long-term goal of this research involves development of a structural energetic model to explain the normal and aberrant IL-7 signaling mechanisms and the generation of new therapeutic peptides/proteins to treat the many deficiencies and diseases associated with impaired IL-7 signaling. Public Health Relevance Statement: This research proposal will focus on the structural, energetic, and cell biological studies of the interaction between human interleukin-7 and its receptor, interleukin-7 receptor alpha. Our understanding of the binding determinants for the IL-7-receptor interaction will be used to engineer novel peptide and protein antagonists using rational protein design and phage display techniques. The long-term goal of this research involves development of a structural energetic model to explain the normal and aberrant IL-7 signaling mechanisms and the generation of new therapeutic peptides/proteins to treat the many deficiencies and diseases associated with impaired human IL-7 signaling.