Structure-function studies on IL-18, IL-18 binding proteins and receptors

说明(申请人提供)：白介素18(IL-18)是一种促炎细胞因子，属于白介素1(IL-1)超家族。它在宿主对微生物的防御中发挥着重要作用，但也参与了几种炎症性疾病的发病，包括类风湿性关节炎、感染性休克和克罗恩病。IL-18信号是由其细胞表面与受体(IL-18R)α亚基结合启动的，随后受体β亚基的募集形成三元信号复合体。IL-18活性在体内由一种自然产生的拮抗剂--IL-18结合蛋白(IL-18BP)通过负反馈机制来调节。痘病毒，包括天花(天花)病毒，也表达功能性IL-18BP同源物，以逃避IL-18介导的宿主免疫反应。因此，IL-18、IL-18R和IL-18BP是开发治疗激动剂炎症性或感染性疾病的有吸引力的靶点，其中IL-18活性下调或上调。然而，对于IL-18如何激活其受体以及IL-18BP如何抑制IL-18尚缺乏透彻的了解。我们建议确定IL-18与IL-18R或IL-18BP的各种蛋白质复合物的晶体结构，并根据结构信息进行功能研究。此外，我们将进行基于结构的IL-18变异体的设计，这些变异体既可以作为一种更有效的细胞因子来逃避痘病毒IL-18bps的中和，也可以作为一种受体拮抗剂来阻断IL-18的活性。目的1.探讨IL-18bps中和IL-18的分子机制。目的2.确定IL-18特异性识别IL-18R 1个亚基的分子机制。目的3.探讨IL-18触发IL-18R1和2亚基异二聚化的分子机制。这一应用的目的是通过结合生物物理和生化方法，包括X射线结晶学、表面等离子体共振(SPR)和IL-18生物测定，对一系列IL-18复合体进行详细表征。完成这一目标是实现我们的长期目标的重要的第一步，即了解IL-18如何激活其受体来启动细胞信号传递以及IL-18BP如何调节这一活动。由于靶向蛋白具有重要的医学意义，我们的研究将为设计选择性抑制剂提供一个平台，最终可能开发出针对多种人类疾病的新疗法。公共卫生相关性：我们的贡献有望提供IL-18BP和IL-18R对IL-18的详细分子识别。这一贡献意义重大，因为它将填补我们目前对IL-18激活途径的认识空白，并将为如何调控IL-18活性提供重要线索。它可能有助于开发针对某些自身免疫性和炎症性疾病的治疗方法，开发针对其他传染病和癌症的免疫疗法，以及打击生物恐怖主义。

DESCRIPTION (provided by applicant): Interleukin-18 (IL-18) is a pro-inflammatory cytokine that belongs to the interleukin-1 (IL-1) superfamily. It plays an important role in host defense against microbes but also contributes to pathogenesis of several inflammatory diseases, including rheumatoid arthritis, septic shock and Crohn's disease. IL-18 signaling is initiated by its cell-surface binding to the receptor (IL-18R) alpha subunit, followed by the recruitment of the receptor beta subunit to form a ternary signaling complex. IL-18 activities are regulated in vivo by a naturally occurring antagonist, the IL-18 binding protein (IL-18BP) through a negative feedback mechanism. Poxviruses, including the smallpox (variola) virus, also express functional IL-18BP homologues to evade IL-18-mediated host immune responses. IL-18, IL-18R and IL-18BP are therefore attractive targets for developing therapeutics agonist inflammatory or infectious diseases where down- or up-modulating IL-18 activities is indicated. However, there is a lack of thorough understanding of how IL-18 activates its receptor and how IL-18BP inhibits IL-18. We propose to determine the crystal structures of various protein complexes of IL-18 with IL-18R or IL-18BP and perform functional studies based on the structural information. In addition, we will perform structure-based design of IL-18 variants that may either serve as a more effective cytokine capable of evading the neutralization of poxvirus IL-18BPs, or as a receptor antagonist capable of blocking IL-18 activities. Aim 1. To determine the molecular mechanism by which IL-18BPs neutralize IL-18. Aim 2. To determine the molecular mechanism by which IL-18 specifically recognizes 1 subunit of IL-18R. Aim 3. To determine the molecular mechanism by which IL-18 triggers the hetero-dimerization of IL-18R1 and 2 subunits. The objective of this application is the detailed characterization of a collection of IL-18 complexes by a combination of biophysical and biochemical methods, including x-ray crystallography, Surface Plasmon Resonance (SPR) and IL-18 bioassay. Accomplishing this objective is an important first step for achieving our long-term goal of understanding how IL-18 activates its receptors to initiate cell signaling and how this activity is regulated by IL-18BP.This research obtains/assumes extra dimensions. Since the targeted proteins are of significant medical relevance, our studies will provide a platform for designing selective inhibitors that may ultimately be developed into new therapeutics against a number of human diseases. Public Health Relevance: Our contribution here is expected to provide detailed molecular recognition of IL-18 by IL-18BP and IL-18R. This contribution is significant because it will fill the gap of our current knowledge on IL-18 activation pathway, and will provide important clues on how to modulate IL-18 activity. It may benefit efforts in developing treatments against some autoimmune and inflammatory diseases, in developing immunotherapies against other infectious diseases and cancer and in combating bioterrorism.