Structural Biology of G Protein-Coupled Receptors

描述（由申请人提供）：趋化因子是促炎介质，用于从循环中招募白细胞到感染或损伤部位。趋化因子受体（ChRs）是7种跨膜（7-TM）螺旋含G蛋白偶联受体（gpcr）；许多是治疗炎症和免疫疾病的有效和选择性药物靶点。由于缺乏关于其高分辨率结构和动力学的信息，目前尚未确定碳重化合物的作用机制。在这个项目中，我们将开发新的技术来克服以前阻碍整体膜蛋白结构测定的主要障碍：充分的表达和纯化，洗涤剂溶液中的稳定，以及晶体生长。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均匀ChRs的生产；2)将chr在洗涤剂溶液中溶解、稳定和纯化，并采用脂质立方相电泳进行无洗涤剂纯化；3)求解了两种主要ChR类（CXCR和CCR）的晶体结构，并优化了在脂质立方相中由二维晶体生长三维晶体的新工艺。这个项目是由一个有才华的研究团队进行的，他们有着出色的记录，包括解决两种7-TM受体（感觉视紫红质II和牛视紫红质）的晶体结构。实现我们的特异性目标将有助于阐明ChRs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、选择性的新型抗炎和免疫调节药物奠定基础。健康相关性：异常趋化因子的作用在炎症性疾病如关节炎和自身免疫性疾病如狼疮中也起着重要作用，而对伤口愈合和抵抗感染的免疫反应至关重要。趋化因子通过特定的细胞表面受体起作用，这些受体在结构上很难表征。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，或抑制免疫系统以防止自身免疫性疾病（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.