Assembly and Control of the Complement Membrane Attack Complex

补体膜攻击复合体的组装和控制

• 批准号: 7356051
• 负责人: Ronald T Ogata
• 金额: $ 30.94万
• 依托单位: TORREY PINES INST FOR MOLECULAR STUDIES
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-03-01 至 2011-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7356051
• 关键词: Active Sites; Affinity; Atherosclerosis; Bacteria; Binding; Biochemical; Biological; Blood Vessels; Cell membrane; Cell surface; Cells; Collaborations; Complement; Complement Membrane Attack Complex; Complex; Complications of Diabetes Mellitus; Cytoplasm; Drug Controls; Environment; Escherichia coli; Goals; Individual; Infection; Inflammatory; Injury; Invaded; Kinetics; Maps; Measures; Membrane; Methods; Modeling; Monitor; Names; Netherlands; Numbers; Object Attachment; Pathogenesis; Process; Protein Binding; Proteins; Protocols documentation; Puncture procedure; Recombinants; Regulation; Relaxation; Research; Resolution; Scotland; Serum Proteins; Site-Directed Mutagenesis; Structure; Surface; Surface Plasmon Resonance; System; Technology; Testing; Universities; Vitronectin; X-Ray Crystallography; assault; complement C5b; complement C5b-6 complex; complement C5b-7 complex; complement C5b-8 complex; design; disulfide bond; domain mapping; driving force; genetic regulatory protein; human disease; inhibitor/antagonist; injured; insight; instrument; killings; molecular modeling; mutant; protein protein interaction; self assembly; sensor; stoichiometry; structural biology; three dimensional structure; tool

DESCRIPTION (provided by applicant): The signature feature of mammalian complement is the ability to kill invading cells by puncturing their plasma membranes. This assault is carried out by the Membrane Attack Complex (MAC), which is a >1,000,000 Da protein complex composed of 5 different serum proteins: single copies of complement components C5b, C6, C7, and C8, and 1 or more copies of component C9. MAC assembly begins with the binding of newly generated C5b to C6 near the target cell surface. Progressively larger complexes are then formed by the spontaneous and sequential addition of C7, C8, and finally multiple copies of C9. Very little is known about this self-assembly process, the structure of the fully-assembled MAC or its intermediates, or even of the 3D structures of the individual MAC proteins. The long-term goal of the proposed studies is to obtain a better picture of the MAC: how the individual proteins are arranged in the complex, what forces drive independent soluble serum proteins to aggregate into the membrane-bound MAC, and how this process is regulated. Our Specific Aims in this application are to (1) use surface plasmon resonance to describe in detail the binding process at each step in MAC assembly and in binding of the regulatory S-protein, (2) map domain-domain interactions early in MAC assembly by expressing individual domains of C6 and C7 in bacteria and measuring their binding activities, (3) determine the 3D structures of expressed domains by NMR or by X-ray crystallography in collaboration with experts in these technologies, and (4) map the active sites within modules by site-specific mutagenesis. These studies should provide insights into the structures of C6 and C7, and into the protein-protein interactions that take place early in MAC formation. This information should allow us to begin to fashion a molecular model of MAC assembly and structure. These studies have substantial relevance to human disease. While the MAC is best known for its defense against infection by foreign cells, it can attack and injure host cells as well. For example, MAC attack on host cells has been identified as a major contributor to the vascular complications of diabetes and the inflammatory pathogenesis of atherosclerosis. A clearer picture of the MAC will provide insights into the design of drugs that control MAC assembly and injury in these prevalent and devastating human diseases.

描述(申请人提供)：哺乳动物补体的标志性特征是通过刺穿入侵细胞的质膜来杀死它们的能力。这种攻击是由膜攻击复合体(MAC)执行的，它是一个100万Da的蛋白质复合体，由5种不同的血清蛋白组成：补体成分C5b、C6、C7和C8的单一拷贝，以及成分C9的1个或多个拷贝。MAC组装开始于新生成的C5b与靶细胞表面附近的C6结合。C7、C8和C9的多个拷贝的自发和顺序相加形成了逐渐变大的复合体。关于这种自组装过程，完全组装的MAC或其中间体的结构，甚至单个MAC蛋白质的3D结构，人们知之甚少。拟议研究的长期目标是更好地了解MAC：单个蛋白质是如何在复合体中排列的，是什么驱使独立的可溶性血清蛋白质聚集到膜结合的MAC中，以及这一过程是如何调控的。我们在这项应用中的具体目标是(1)使用表面等离子共振详细描述MAC组装中每一步的结合过程以及调控的S-蛋白质的结合；(2)通过在细菌中表达C6和C7的单个结构域并测量其结合活性来映射MAC组装早期的结构域-结构域相互作用；(3)通过核磁共振或与这些技术专家合作通过X射线结晶学确定表达结构域的三维结构；以及(4)通过定点突变定位模块内的活性位点。这些研究应该对C6和C7的结构以及在MAC形成早期发生的蛋白质-蛋白质相互作用提供洞察力。这些信息应该可以让我们开始建立MAC组装和结构的分子模型。这些研究与人类疾病有实质性的相关性。虽然MAC最为人所知的是防御外来细胞的感染，但它也可以攻击和伤害宿主细胞。例如，对宿主细胞的MAC攻击已被确认为糖尿病血管并发症和动脉粥样硬化炎症发病机制的主要贡献者。对MAC的更清晰的了解将为药物的设计提供见解，这些药物可以控制MAC的组装和在这些普遍和毁灭性的人类疾病中的损伤。