Structure, function and engineering of immune cytokine receptor signaling

描述（由申请人提供）：免疫调节细胞因子与跨膜信号受体结合，以介导广泛的功能，包括白细胞增殖、分化和扩增。大多数免疫调节细胞因子具有对正常免疫稳态至关重要的冗余和独特的活性，但这种功能多效性为这些细胞因子作为药物的有效靶向使用提出了主要问题。细胞因子多效性是少量共享受体（例如共同γ链和gp 130）接合许多不同细胞因子的结果，所述细胞因子然后通过有限的JAK和STAT蛋白质组激活重叠的细胞内信号传导途径。在该奖项的前一个学期，我们获得了一系列具有共享受体的不同细胞因子复合物的细胞外结构架构的赞赏，包括IL-1，IL-2，IL-4，IL-6，IL-13，IL-17，IL-23和IFN的细胞因子复合物，其表现出异二聚体信号传导几何结构的惊人多样性。在此更新申请中，我们将研究重点放在多效性细胞因子IL-2和IL-15上，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传导、JAK和STAT的细胞内活化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传导特异性中起作用，以及通过基于结构的受体相互作用的细胞因子工程“调谐”信号传导是否是开发具有增强功效、细胞亚群偏好和降低毒性的新型免疫治疗剂的可行手段。这个高度合作的建议的总体目标是：目的1-确定的生物物理基础的功能冗余和特异性表现出两个“天然替代品”？c细胞因子，IL-2和IL-15，通过共享的信号受体（IL-2 R和？目的2-利用基于结构的蛋白质工程来尝试产生具有不同信号传导特性和T细胞亚群偏好的IL- 2变体，其可能是更有效的免疫治疗剂，如合作者在多种体内疾病模型中评估的;目的3-确定抗体增强IL-2对不同T细胞亚群的活性的机制基础，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，在目标4中，我们继续 通过重建与细胞因子和细胞内JAK结合的完整全长细胞因子受体跨膜复合物，通过晶体学和电子显微镜成像，来寻求关于细胞因子受体细胞内片段如何接合Janus激酶（JAK）分子的结构信息。 以这种方式，通过结合结构（X射线晶体学，电子显微镜，和NMR），蛋白质工程，信号，和体内研究，我们建议获得一个完整的分子快照共享的细胞因子受体信号从配体的初始接合通过细胞内信号级联的激活。

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.