Structure/Function of Terminal Complement Proteins

描述(申请人提供)：这个项目的长期目标是了解人类C5b-9的形成和功能的分子细节，C5b-9是补体的细胞溶解复合体，称为“膜攻击复合体”或MAC。Mac由C5b、C6、C7、C8和C9组成。在这些成分中，C6、C7、C8α和C8β亚基以及C9是同源的，而C8Gamma则是补体系统中唯一的Lipocalin。这个项目将把C8作为一个模型系统来识别MAC蛋白中的结构-功能关系。重点将放在C8Gamma和它在MAC的形成和功能中具有以前未被认识到的作用的可能性上。具体目标是：(1)表征C8Gamma的配体结合性质。C8Gamma晶体结构显示典型的Lipocalin折叠，与未知小分子有明显的结合位置。研究将集中在脂肪酸和相关化合物作为潜在的配体，以确定C8Gamma是否具有与膜脂结合的能力，例如甘油磷脂和脂多糖，或者可能是释放在MAC附近的可溶性促炎分子；(2)检验C8Gamma通过与膜相关脂直接相互作用来增强MAC活性的假设。将制备限制配体结合的突变体，并对其保留/丧失增强MAC溶血和杀菌活性的能力进行表征。阳性结果提示C8Gamma可能参与了在有核细胞中诱导MAC介导的反应；(3)确定C8中重要的功能结合部位。将使用C8Gamma及其结构同系物NGAL的嵌合结构来确定C8Gamma上与C8pha结合的位点，并测试这些嵌合结构是否与C8pha结合。与中间MAC复合体C5b-7结合的C8beta区域将使用C8pha/C8beta嵌合体和C8beta缺失突变体来确定模块的作用；(4)生产用于结晶和结构研究的MAC蛋白片段。C8α和C8β的片段将重组产生，并通过对血清来源的C8进行蛋白质分解消化而产生。前一种方法将强调中心MACPF片段，因为它很可能是可以表达的自折叠结构域。后一种方法将利用规模优势。完成后，结果将为MAC蛋白相互作用和目标细胞膜的机制提供新的见解。这些信息将有助于设计和开发治疗上有用的MAC类似物以及MAC裂解和刺激功能的调节因子。由于MAC蛋白及其功能的独特性，所获得的信息将有助于从总体上理解蛋白质结构与功能的关系。

DESCRIPTION (provided by applicant): The long-term objective of this project is to understand molecular details of the formation and function of human C5b-9, the cytolytic complex of complement referred to as the "membrane attack complex" or MAC. MAC is composed of C5b, C6, C7, C8 and C9. Among these components, C6, C7, the C8alpha and C8beta subunits, and C9 are homologous whereas C8gamma has the distinction of being the only lipocalin in the complement system. This project will focus on C8 as a model system to identify structure-function relationships within the MAC proteins. Emphasis will be on C8gamma and the possibility it has a previously unrecognized role in MAC formation and function. Specific aims are: (1) to characterize the ligand binding properties of C8gamma. The C8gamma crystal structure displays a typical lipocalin fold with a distinct binding site for an as yet unidentified small molecule. Studies will focus on fatty acids and related compounds as potential ligands to determine if C8gamma has the capacity to bind membrane lipid, e.g. glycerophospholipids and LPS, or possibly soluble proinflammatory molecules released in the vicinity of the MAC; (2) to test the hypothesis that C8gamma enhances MAC activity through direct interaction with membrane-associated lipid. Mutants in which ligand binding is restricted will be prepared and characterized with respect to retention/loss of ability to enhance MAC hemolytic and bactericidal activities. Positive results would suggest C8gamma may be involved in inducing MAC-mediated responses in nucleated cells; (3) to identify functionally important binding sites in C8. The site on C8gamma that binds C8alpha will be identified using chimeric constructs of C8gamma and its structural homologue NGAL, and testing these for C8alpha binding. The region of C8beta which binds the intermediate MAC complex C5b-7 will use C8alpha / C8beta chimeras and C8beta deletion mutants to determine the role of the modules; (4) to produce fragments of the MAC proteins for crystallization and structural studies. Segments of C8alpha and C8beta will be produced recombinantly and by proteolytic digestion of serum-derived C8. The former approach will emphasize the central MACPF segment because it is likely to be self-folding domain that can be expressed. The latter approach will take advantage of scale. When complete, results will provide new insight into mechanisms by which the MAC proteins interact with each other and the target cell membrane. Such information will facilitate design and development of therapeutically useful analogues of MAC and regulators of MAC lytic and stimulatory functions. Because of the uniqueness of the MAC proteins and their function, information obtained will contribute to an understanding of protein structure-function relationships in general.