STRUCTURE & FUNCTION OF TERMINAL COMPLEMENT PROTEINS

这个项目主要关注的是分子细节的组装和功能 人C5b-9，末端补体蛋白C5b的细胞溶解复合体， C6、C7、C8和C9。重点将放在结构的特征上， 人类C8的功能和分子遗传学以了解其作用 在C5b-9的形成和功能中，其高度特殊的意义 结构、其与其他终端组件的进化关系以及 C8分子缺陷在蛋白质和蛋白质两方面的性质 和基因水平。要追求的具体目标包括将 编码人类C8的α、β和伽马亚基的基因。重点 将进行染色体分配，确定基因大小，识别 物理链接，基因组克隆作图，确定外显子 组织，描述可能的监管区域并确定 进化关系。结果将为以下工作奠定基础 C8缺陷症的基因缺陷分析及对C8的未来研究 监管。结构-功能研究将确定阿尔法的片段， 参与β-α和α-C9相互作用的β和C9。 实验将探索守恒结构的可能性 这些同源蛋白中的结构域介导β-α-C9共... 溶液中的聚合，并且这种相互作用可以构成 C5b-9组装的潜在机制。相关实验将重点放在 C5-β和α-伽马，以进一步了解 终端组件之间的识别。要使用的方法包括 蛋白质片段、功能结构域的分离和化学成分 修改。在其他实验中，阿尔法和贝塔的片段 直接与靶膜相互作用将被放射性标记 光敏、膜限制探针，隔离、表征和 与那些预测为膜相互作用的片段相比， 氨基酸序列。这些部分在Tact C8中的可访问性将是 评估以测试此类数据段不可访问的假设 在与C5b-7联系之前。实验还将探索 C5b-9结构类似物作为细胞溶血剂的用途。共轭化合物 针对细胞表面抗原的Fab-C8将进行测试 因为它们有能力调节C9结合并产生膜损伤。是这样的 类似物可能作为治疗剂有用。了解蛋白质- C5b-9组装过程中的蛋白质和蛋白质-膜相互作用 普遍的生物学重要性，因为它是一种独特的系统 作为细胞杀伤的内源性媒介发挥作用。

This project focuses on molecular details of the assembly and function of human C5b-9, the cytolytic complex of terminal complement proteins C5b, C6, C7, C8 and C9. Emphasis will be on characterizing the structure, function and molecular genetics of human C8 so as to understand its role in C5b-9 formation and function, the significance of its highly peculiar structure, its evolutionary relationship to other terminal components and the nature of the molecular defect in C8 deficiencies at both the protein and gene levels. Specific aims to be pursued include characterizing the genes encoding the alpha, beta, and gamma subunits of human C8. Emphasis will be on chromosomal assignments, determining gene size, identifying physical linkages, mapping of genomic clones, determining exonintron organization, characterizing possible regulatory regions and identifying evolutionary relationships. Results will provide the foundation for analysis of gene defects in C8 deficiencies and for future studies of C8 regulation. Structure-function studies will identify segments in alpha, beta and C9 that are involved in beta-alpha and alpha-C9 interactions. Experiments will explore the possibility that conserved structural domains in these homologous proteins mediate beta-alpha-C9 co- polymerization in solution and that such interactions may constitute the underlying mechanism of C5b-9 assembly. Related experiments will focus on C5-beta and alpha-gamma in order to further understand the basis for recognition amongst terminal components. Approaches to be used include protein fragmentation, isolation of functional domains and chemical modifications. In other experiments, segments of alpha and beta that interact directly with target membranes will be radiolabeled by photosensitive, membrane-restricted probes, isolated, characterized and compared to those segments predicted to be membrane-interacting based on amino acid sequence. Accessibility of these segments in tact C8 will be evaluated to test the hypothesis that such segments are inaccessible prior to association with C5b-7. Experiments will also explore the utility of structural analogues of C5b-9 as cytolytic agents. Conjugates of Fab-C8 that are directed against cell-surface antigens will be tested for their ability to mediate C9 binding and produce membrane damage. Such analogues could be useful as therapeutic agents. Understanding protein- protein and protein-membrane interactions during C5b-9 assembly is of general biological importance because it is a unique system that functions as an endogenous mediator of cell killing.