Assembly and Control of the Complement Membrane Attack Complex

描述（由申请人提供）：哺乳动物补体的特征是通过刺穿侵入细胞的质膜来杀死侵入细胞的能力。这种攻击是由膜攻击复合物（MAC）进行的，MAC是一种由5种不同血清蛋白组成的> 1，000，000 Da蛋白质复合物：补体成分C5 b，C6，C7和C8的单拷贝，以及成分C9的1个或多个拷贝。MAC组装开始于新产生的C5 b与靶细胞表面附近的C6结合。然后通过自发和顺序添加C7，C8，最后是C9的多个拷贝形成逐渐更大的复合物。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蛋白的3D结构，我们知之甚少。拟议研究的长期目标是更好地了解MAC：单个蛋白质如何在复合物中排列，是什么力量驱动独立的可溶性血清蛋白聚集成膜结合MAC，以及如何调节这一过程。我们在本申请中的具体目的是（1）使用表面等离子体共振来详细描述MAC组装中每个步骤的结合过程和调节S-蛋白的结合，（2）通过在细菌中表达C6和C7的各个结构域并测量它们的结合活性来绘制MAC组装早期的结构域-结构域相互作用，（3）通过NMR或X射线晶体学与这些技术的专家合作确定表达结构域的3D结构，以及（4）通过位点特异性诱变绘制模块内的活性位点。这些研究应该提供深入了解C6和C7的结构，以及MAC形成早期发生的蛋白质-蛋白质相互作用。这些信息将使我们开始建立MAC组装和结构的分子模型。这些研究与人类疾病有很大的相关性。虽然MAC以其抵抗外来细胞感染而闻名，但它也可以攻击和伤害宿主细胞。例如，MAC对宿主细胞的攻击已被鉴定为糖尿病的血管并发症和动脉粥样硬化的炎性发病机制的主要贡献者。MAC的更清晰的图片将提供深入了解药物的设计，控制MAC组装和损伤，在这些流行的和毁灭性的人类疾病。

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.