MCP--STRUCTURE, MICROBIAL INTERACTIONS AND FUNCTION

MCP--结构、微生物相互作用和功能

• 批准号: 6704229
• 负责人: John Atkinson
• 金额: $ 34.65万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-04-01 至 2006-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6704229
• 关键词: Neisseriaceae; Streptococcus pyogenes; X ray crystallography; active sites; biological signal transduction; complement inhibitors; complement pathway; decay accelerating factor; fertility; genetically modified animals; laboratory mouse; nuclear magnetic resonance spectroscopy; phosphorylation; protein structure function; protein tyrosine kinase; tissue /cell culture; yeast two hybrid system

DESCRIPTION (adapted from investigator's abstract): The complement system is ingeniously designed to prevent infections as well as to process immune complexes and damaged tissue. Strict control of its activation during innate and acquired humoral immune responses is critical to minimize damage to host tissue. Membrane cofactor protein (MCP; CD46) is widely expressed inhibitor of complement activation at the critical step of C3/C5 convertase generation. It serves as a cofactor for the serine protease factor I to cleave and thereby inactivate C3b and C4b that deposit on host tissue. Most cells and tissues express MCP as a family of four isoforms that differ in their O-glycosylation and cytoplasmic tails. MCP is a receptor for three human pathogens: measles virus, Streptococcus pyogenes and Neisseria. Attachment of the measles virus of Neisseria to MCP transmits signals for IL-12 down-regulation in monocytes or for calcium fluxes in epithelial cells, respectively. MCP has also been implicated in reproduction, in large part due to its dense expression on placental trophoblast and on the inner acrosome membrane of spermatozoa. Because of its potent complement regulator activity, MCP has been recombinantly produced for use as a soluble therapeutic agent and engineered into pigs whose organs are being employed for xenografting. In this grant application, we propose to continue our studies on the structure, microbial interactions and function of MCP. We postulated and later demonstrated during the prior grant period that each of four regularly expressed isoforms of MCP possesses functional advantages. In this renewal application we continue this focus while placing an increased emphasis on microbial connections and cell signaling. The active sites of MCP will be characterized by NMR spectroscopy and X-ray crystallography. We have recently demonstrated that in three cell types MCP is tyrosine phosphorylated on one of its two cytoplasmic tails. For this signaling event, we propose a systematic analysis of the site(s), responsible kinase(s), and related downstream events. Additionally, we will explore in depth the microbial interactions with MCP as they relate to binding sites, signaling events, and three-dimensional structure. We will characterize three strains of transgenic mice expressing human MCP. We anticipate these animals will be a valuable tissue source. We also propose a targeted disruption of the MCP mouse gene since MCP is expressed predominantly on the inner acrosomal membrane of mouse spermatozoa. The fertility of these mice will be assessed and they will be crossed with other deficient mice strains to explore the role of MCP in reproduction. Lastly, the mechanism of action of MCP in situ will be analyzed by quantitative methods developed during the prior granting period. We will address such questions as the role of membrane versus fluid phase inhibitors and contribution of MCP versus decay accelerating factor. The specific aims of this proposal outline a broad-based approach to increase our understanding of complement regulation by one if its major inhibitory proteins. Additionally, these experiments will expand our knowledge of the fascinating interactions of this complement regulator with three common infectious diseases and its role in cell signaling events and reproduction.

描述（根据调查员的摘要改编）：补充系统是 巧妙地设计以防止感染和处理免疫 复合物和组织受损的组织。严格控制其先天性的激活 并且获得的体液免疫反应对于最大程度地减少对宿主的损害至关重要 组织。膜辅因子蛋白（MCP; CD46）广泛表达的抑制剂 在C3/C5转化酶产生的关键步骤中补体激活。它 用作丝氨酸蛋白酶因子I裂解的辅因子，从而 使沉积在宿主组织上的C3B和C4B失活。大多数细胞和组织 Express MCP作为四个同工型家族，其O-糖基化不同 和细胞质尾巴。 MCP是三种人类病原体的受体：麻疹 病毒，化脓性链球菌和奈瑟氏菌。麻疹病毒的附着 到MCP的奈瑟氏菌在单核细胞中传输IL-12下调的信号 分别用于上皮细胞中的钙通量。 MCP也是 与繁殖有关，在很大程度上是由于其密集的表达 胎盘滋养细胞和精子的内部缩小膜上。 由于其有效的补体调节活性，MCP已重组 用于用作可溶性治疗剂并设计成猪的生产 正在使用器官进行异种。在此赠款申请中，我们 建议继续我们对结构，微生物相互作用和 MCP的功能。我们假设并后来在先前的赠款中证明 四个定期表达的MCP同工型的时期 功能优势。在此续订应用程序中，我们继续关注此重点 越来越重视微生物连接和细胞信号传导。这 MCP的活动位点将以NMR光谱和X射线为特征 晶体学。我们最近证明，在三种细胞类型中，MCP是 酪氨酸在其两个细胞质尾部之一上磷酸化。对于此信号 事件，我们提出了对位点，负责激酶的系统分析， 和相关的下游事件。此外，我们将深入探索 与MCP相关的微生物相互作用与结合位点相关，信号传导 事件和三维结构。我们将表征三种菌株 表达人MCP的转基因小鼠。我们预计这些动物将是 有价值的组织来源。我们还提出了MCP鼠标的目标破坏 基因由于MCP主要表达 鼠标精子。这些小鼠的生育能力将被评估，它们将 与其他缺陷的小鼠菌株交叉以探索MCP的作用 生殖。最后，将分析MCP原位作用机理 通过先前授予期间开发的定量方法。我们将 解决诸如膜与液相抑制剂的作用之类的问题 MCP与衰减加速因子的贡献。具体目的 该建议概述了一种基于广泛的方法，以提高我们对 如果其主要的抑制蛋白补充调节。此外， 这些实验将扩大我们对令人着迷的互动的了解 这种补充调节子具有三种常见的传染病及其在 细胞信号事件和繁殖。