Structure, function and engineering of immune cytokine receptor signaling

免疫细胞因子受体信号传导的结构、功能和工程

• 批准号: 9297185
• 负责人: Kenan Christopher GARCIA
• 金额: $ 39.25万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-04-01 至 2018-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9297185
• 关键词: Address; Affinity; Antibodies; Architecture; Autoimmunity; Award; Binding; Biological; Biology; Biophysics; Cells; Chemistry; Chemosensitization; Complement; Complex; Cryoelectron Microscopy; Crystallography; Cytokine Receptors; Cytokine Signaling; Development; Diabetes Mellitus; Dimerization; Disease model; Electron Microscopy; Engineering; Evolution; Exhibits; Experimental Autoimmune Encephalomyelitis; Extracellular Structure; Gap Junctions; Gene Expression; Geometry; Goals; Grant; Homeostasis; Human; IL6ST gene; Image; Immune; Immune System Diseases; Immunotherapeutic agent; Immunotherapy; In Vitro; Instruction; Interferons; Interleukin 2 Receptor; Interleukin 2 Receptor Gamma; Interleukin 6 Receptor; Interleukin-1; Interleukin-13; Interleukin-15; Interleukin-17; Interleukin-2; Interleukin-4; Interleukin-6; Janus kinase; Knowledge; Left; Length; Ligand Binding; Ligands; Link; Malignant Neoplasms; Mediating; Molecular; Molecular Conformation; Molecular Profiling; Outcome; Paint; Pharmaceutical Preparations; Play; Privatization; Property; Protein Engineering; Proto-Oncogene Proteins c-akt; Receptor Signaling; Regulatory T-Lymphocyte; Reporting; Resolution; Role; STAT protein; Signal Pathway; Signal Transduction; Specificity; Stat5 protein; Structural Protein; Structure; Structure-Activity Relationship; System; T-Lymphocyte Subsets; Techniques; Testing; Therapeutic; Time; Toxic effect; Translating; Variant; X-Ray Crystallography; base; cell type; combinatorial; conformer; cytokine; cytokine receptor gp130; design; dimer; extracellular; functional outcomes; graft vs host disease; human disease; immune system function; immunoregulation; improved; in vivo; in vivo Model; insight; interleukin-15 receptor; interleukin-23; interleukin-6 receptor alpha; leukocyte proliferation; melanoma; network dysfunction; novel; pleiotropism; preference; public health relevance; receptor; receptor binding; reconstitution; reconstruction; structural biology

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" γc cytokines, IL-2 and IL-15, that act through shared signaling receptors (IL-2Rß and γc) 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.

说明(申请人提供)：免疫调节细胞因子与跨膜信号受体结合，以调节广泛的功能，包括白细胞的增殖、分化和扩增。大多数免疫调节细胞因子都具有冗余和独特的活性，这些活性对正常的免疫动态平衡至关重要，但这种功能多效性是这些细胞因子作为药物有效、有针对性地使用的主要问题。细胞因子的多效性是少数共同的受体，如共同的伽马链和gp130，结合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的结果。在该奖项的前一期，我们获得了对具有共同受体的不同细胞因子复合体的胞外结构的赞赏，包括IL-1、IL-2、IL-4、IL-6、IL-13、IL-17、IL-23和干扰素的复合体，它们展示了惊人的异二聚体信号几何结构的多样性。在这一新的应用中，我们将重点研究多效性细胞因子IL-2和IL-15，以探讨受体-细胞因子复合体的细胞外结构如何影响跨膜信号、JAK和STAT的细胞内激活，以及随后的体内功能。我们希望确定IL-2和IL-15受体复合体亚单位的结合化学和几何结构是否在调节信号特异性方面发挥作用，以及通过基于结构的细胞因子工程受体相互作用来调节信号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。这项高度协作的提案的总体目标是：目的1-确定两种“天然替代”γc细胞因子IL-2和IL-15所表现出的功能冗余和特异性的生物物理基础，这两种细胞因子通过共享的信号受体(IL-2Rü和γc)而作用于私有的α受体；目的2-利用基于结构的蛋白质工程试图创造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，如合作者在各种体内疾病模型中所评估的那样；目的3-确定抗体增强IL-2对不同T细胞亚群活性的机制，并发现能够重塑野生型IL-2构象并改变其生物学活性的新的增强抗体。最后，在目标4中，我们继续探索细胞因子受体细胞内片段如何与Janus Kinase(JAK)分子结合的结构信息，方法是重组整个全长细胞因子受体跨膜复合体，结合到细胞因子和细胞内JAK上，通过结晶学和电子显微镜进行成像。通过这种方式，通过结合结构(X射线结晶学、电子显微镜和核磁共振)、蛋白质工程、信号和体内研究，我们建议通过激活细胞内信号级联来获得从配体的初始参与到共享的细胞因子受体信号的完整的分子快照。