Structural Biology of G Protein-Coupled Receptors

描述（由申请方提供）：趋化因子是促炎介质，用于将白细胞从循环中募集到感染或损伤部位。趋化因子受体（ChR）是含有七个跨膜（7-TM）螺旋的G蛋白偶联受体（GPCR）;许多是用于治疗炎性和免疫病症的有效和选择性药物靶标。ChRs的作用机制尚未确定，主要原因是缺乏关于其高分辨率结构和动态的信息。在这个项目中，我们将开发新的技术，以克服主要的障碍，以前排除了结构确定的完整的膜蛋白：充分的表达和纯化，在洗涤剂溶液中的稳定，晶体生长。我们的主要目标是阐明ChRs的晶体结构。我们的具体目标是：1）通过一种新的腺病毒介导的小鼠肝脏表达系统，优化适合晶体生长的同质ChR的生产; 2）在去污剂溶液中溶解、稳定和纯化ChR，并采用基于无去污剂的无规立方相电泳进行纯化; 3）解决了两种主要ChR类（CXCR和CCR）中的每一种的代表的晶体结构，并优化了用于从在无规立方相中的2-D晶体生长3-D晶体的新程序。这个项目由一个有才华的研究团队进行，他们有着出色的记录，包括解决两个7-TM受体（感觉视紫红质II和牛视紫红质）的晶体结构。实现我们的特定目标将有助于阐明ChR的作用机制，并为合理设计针对特定受体状态独特位点的高效和选择性的新型抗炎和免疫调节药物奠定基础。健康相关性：虽然对于适当的伤口愈合和免疫反应对抗感染至关重要，但异常趋化因子作用也在诸如关节炎和狼疮等自身免疫疾病中起作用。趋化因子通过特定的细胞表面受体起作用，这些受体在结构上非常难以表征。我们的项目旨在克服这种表征的障碍，从而为开发药物奠定基础，这些药物可以在我们面临危及生命的感染时增强我们的免疫系统，或者抑制它以防止自身免疫性疾病（例如狼疮）或移植器官排斥引起的组织损伤。

DESCRIPTION (provided by applicant): Chemokines are pro-inflammatory mediators for the recruitment of leukocytes from the circulation to the site of an infection or injury. Chemokine receptors (ChRs) are seven transmembrane (7-TM) helix- containing G protein-coupled receptors (GPCRs); many are effective and selective drug targets for the treatment of inflammatory and immune disorders. The mechanisms of action of ChRs are not yet defined, due largely to a lack of information on their high-resolution structures and dynamics. In this project we will develop novel technologies to overcome the major hurdles that previously precluded structural determination of integral membrane proteins: adequate expression and purification, stabilization in detergent solutions, and crystal growth. Our major goal is to elucidate the ChRs' crystal structures. Our Specific Aims are to 1) optimize the production of homogeneous ChRs suitable for crystal growth, via a novel adenovirus-mediated mouse liver expression system; 2) solubilize, stabilize and purify the ChRs in detergent solutions, and adapt lipidic cubic phase-based electrophoresis for their detergent-free purification; 3) solve the crystal structures of one representative of each of the two major ChR classes (CXCR and CCR), and optimize a novel procedure for growing 3-D crystals from 2-D crystals in the lipidic cubic phase. This project is being conducted by a talented team of researchers with an excellent track record, including solving the crystal structures of two 7-TM receptors (sensory rhodopsin II & bovine rhodopsin). Achieving our Specific Aims will help elucidate the ChRs' mechanism of action, and lay the foundation for rational design of highly potent and selective new anti-inflammatory and immunomodulatory drugs targeting sites unique to specific receptor states. Health Relevance: While vital for proper wound healing and immune responses to fight infections, abnormal chemokine action also plays a role in such inflammatory diseases as arthritis and auto-immune disease as Lupus. Chemokines act via specific cell surface receptors which have been extremely difficult to characterize structurally. Our project seeks to overcome the obstacles to such characterization and so lay the foundation for developing drugs that could boost our immune system when we are faced with a life- threatening infection, or to dampen it to prevent tissue damage caused by autoimmune disorders (e.g. Lupus) or rejection of transplanted organs.