Assembly and Control of the Complement Membrane Attack Complex

描述（由申请人提供）：哺乳动物补体的标志性特征是能够通过刺穿其质膜来杀死入侵细胞。这种攻击是由膜攻击复合体（MAC）进行的，这是一个由5种不同的血清蛋白组成的bb100万Da蛋白质复合体：补体组分C5b、C6、C7和C8的单拷贝，以及组分C9的1个或多个拷贝。MAC组装开始于新生成的C5b与目标细胞表面附近的C6结合。随后，C7、C8和C9的多个副本自发和顺序的加入，逐渐形成更大的复合物。对于这种自组装过程，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，我们所知甚少。提出的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，是什么力量驱使独立的可溶性血清蛋白聚集到膜结合的MAC中，以及这个过程是如何调节的。我们在这个应用程序中的具体目标是：(1)使用表面等离子体共振来详细描述MAC组装和调节s蛋白结合的每一步的结合过程，(2)通过表达细菌中C6和C7的单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)与这些技术的专家合作，通过核磁共振或x射线晶体学确定表达结构域的3D结构。(4)通过位点特异性诱变绘制模块内的活性位点。这些研究将有助于深入了解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。这一信息应该允许我们开始塑造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最著名的功能是抵御外来细胞的感染，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确定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因素。更清晰的MAC图谱将为控制这些流行和毁灭性人类疾病中MAC组装和损伤的药物设计提供见解。

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.