Structure, function and engineering of immune cytokine receptor signaling

描述（由申请人提供）：免疫调节细胞因子参与跨膜信号受体，以介导包括白细胞增殖，分化和扩张在内的广泛功能。大多数免疫调节细胞因子都具有对正常免疫稳态至关重要的冗余和独特的活性，但这种功能上的多效性为这些细胞因子作为药物的有效、靶向使用提出了一个主要问题。细胞因子多效性是少数共享受体的结果，如公共γ链和gp130，参与许多不同的细胞因子，然后通过一组有限的JAK和STAT蛋白激活重叠的细胞内信号通路。在此奖项的前一个任期内，我们获得了对具有共享受体的不同细胞因子复合物光谱的细胞外结构结构的赞赏，包括IL-1， IL-2, IL-4, IL-6, IL-13, IL-17， IL-23和IFN，它们表现出惊人的异二聚体信号几何形状的多样性。在这项更新申请中，我们将重点研究多效性细胞因子IL-2和IL-15，以了解受体-细胞因子复合物的细胞外结构如何影响跨膜信号，细胞内JAK和STAT的激活以及随后的体内功能。我们希望确定IL-2和IL-15受体复合物亚基的结合化学和几何结构是否在调节信号特异性中发挥作用，以及通过基于结构的受体相互作用的细胞因子工程“调节”信号传导是否是开发具有增强疗效，细胞亚群偏好和降低毒性的新型免疫疗法的可行手段。这个高度协作的建议的总体目标是：目标1-确定两个“天然替代品”所表现出的功能冗余和特异性的生物物理基础？c细胞因子，IL-2和IL-15，通过共享信号受体(IL-2R¿和？C)但私人α受体；目的2-利用基于结构的蛋白质工程来尝试创建具有不同信号特性和T细胞亚群偏好的IL- 2变体，这可能是更有效的免疫疗法，正如合作者在各种体内疾病模型中评估的那样；和Aim 3-确定针对不同T细胞亚群的抗体增强IL-2活性的机制基础，以及发现新的增强抗体，可以重塑野生型IL-2的构象并改变其生物活性。最后，在Aim 4中，我们继续

DESCRIPTION (provided by applicant): Immunoregulatory cytokines engage transmembrane signaling receptors in order to mediate a wide range of functions including leukocyte proliferation, differentiation, and expansion. Most immunoregulatory cytokines possess both redundant and distinct activities that are critical to normal immune homeostasis, but this functional pleiotropy presents a major problem for the effective, targeted use of these cytokines as drugs. Cytokine pleiotropy is a consequence of a small number of shared receptors, such as common gamma chain and gp130, engaging many different cytokines, which then activate overlapping intracellular signaling pathways through a limited set of JAK and STAT proteins. During the prior term of this award, we gained an appreciation for the extracellular structural architectures of a spectrum of different cytokine complexes with shared receptors, including those of IL-1, IL-2, IL-4, IL-6, IL-13, IL-17, IL-23, and IFN, which exhibited an astonishing diversity of heterodimeric signaling geometries. In this renewal application, we focus our studies on the pleiotropic cytokines IL-2 and IL-15, to ask how extracellular structures of the receptor-cytokine complexes influence transmembrane signaling, intracellular activation of JAK and STAT, and subsequent in vivo function. We wish to determine if the binding chemistry and geometry of the IL-2 and IL-15 receptor complex subunits plays a role in modulating signaling specificity, and whether "tuning" signaling through structure-based cytokine engineering of receptor interactions is a viable means of developing novel immunotherapeutics with enhanced efficacy, cell subset preferences, and reduced toxicity. The overall goals of this highly collaborative proposal are: Aim 1- to determine the biophysical basis for the functional redundancy and specificity exhibited by two "natural surrogate" ?c cytokines, IL-2 and IL-15, that act through shared signaling receptors (IL-2R¿ and ?c) but private alpha-receptors; Aim 2- to utilize structure-based protein engineering to attempt to create IL- 2 variants with diverse signaling properties and T cell subset preferences, that may be more effective immunotherapeutics as assessed by collaborators in a variety of in vivo disease models; and Aim 3- to determine the mechanistic basis of antibody potentiation of IL-2 activity towards distinct T cell subsets, as well as discover new potentiating antibodies that could remodel the conformation of wild-type IL-2 and alter its biological activity. Finally, in Aim 4, we continue to pursue structural information on how cytokine receptor intracellular segments engage Janus Kinase (JAK) molecules, by reconstituting an entire full-length cytokine receptor transmembrane complex, bound to both cytokine and intracellular JAK for imaging by crystallography and electron microscopy. In this fashion, by combining structure (X-ray crystallography, Electron Microscopy, and NMR), protein engineering, signaling, and in vivo studies, we propose to obtain a complete molecular snapshot of shared cytokine receptor signaling from the initial engagement of ligand through the activation of intracellular signaling cascades.