MECHANISM OF COMPLEMENT-MEDIATED MEMBRANE DAMAGE

补体（C）是一组血清蛋白，其构成体液免疫系统。 我们免疫防御系统的手臂。 其多种功能包括裂解 某些细菌、病毒和细胞通过直接攻击细菌， 病毒或细胞膜。 C对膜的攻击涉及到 膜上C末端蛋白（C5 b-9）的多聚体复合物， 插入到膜中以形成可渗透离子的通道， 溶质。 然而，通道形成的机制，这些插入 蛋白质尚未解决。 拟议研究的长期目标是开发一种更清晰的 了解补体介导的细胞溶解机制， 控制补体抗肿瘤功效的调节过程 同源细胞 本课题的具体目标是：1）分析 C5 b-8和C5 b-9复合物的结构/功能关系， 渗透到膜的深度和之间的协同性 单个复合物形成功能通道，2）系统地 探讨温度对钙通道的影响及Ca ~（2+）在通道中的作用 组装和功能，以及3）研究膜的参与 表面分子调节插入，从而调节裂解效率， 同源细胞上的膜结合末端复合物。 的深度 在末端复合物中的单个蛋白质的渗透将是 使用膜限制性光反应糖脂探针测定 锚定在外层或内层单层的特定位置， 模型膜将被用作C攻击的基板。 的 通道形成的动力学将通过比较 插入与标记物从囊泡释放的动力学作为函数 的浓度。 这些方法将使我们能够 评价温度和Ca+2离子对分离的具体影响 过程（例如，结合，插入，C9聚合）参与通道 组装件. 除了影响功能的理化因素外， 组装的末端复合物，膜因素的影响也将 被研究。 特别是，末端蛋白质与 膜表面蛋白质将使用光敏 交联试剂以检测相关蛋白质。 的相关性 这些与末端复合物插入的关联将被 研究了

Complement(C) is a group of serum proteins which constitutes the humoral arm of our immune defense system. Its diverse functions include lysis of certain bacteria, viruses and cells by direct attack on the bacterial, viral or cellular membranes. Membrane attack by C involves the assembly of a multimeric complex of C terminal proteins (C5b-9) on membrane which inserts into the membranes to form channels permeable to ions and small solutes. However, the mechanism of channel formation by these inserted proteins is still unresolved. The long-range goal of the proposed research is to develop a clearer understanding of the mechanism of complement-mediated lysis of cells and of the regulatory processes controlling the efficacy of complement against homologous cells. The specific aims of this project are: 1) to analyze structure/function relationships of the C5b-8 and C5b-9 complexes in terms of depth of penetration into the membrane and cooperativity between individual complexes in forming functional channels, 2) to systematically investigate the effect of temperature and the role of Ca+2 in channel assembly and function, and 3) to investigate the involvement of membrane surface molecules in modulating insertion, and hence lytic efficiency, of membrane-bound terminal complexes on homologous cells. The depth of penetration of the individual proteins in terminal complexes will be determined using membrane-restricted, photo-reactive glycolipid probes anchored at defined positions in either the outer or the inner monolayer of model membranes which will be used as substrates for C attack. The dynamics of channel formation will be studied by comparing the kinetics of insertion with the kinetics of marker release from vesicles as a function of the concentration of inserted complexes. These methods will allow us to evaluate the specific effects of temperature and Ca+2 ions on separate processes (eg. binding, insertion, C9 polymerization) involved in channel assembly. In addition to the physicochemical factors affecting functional assembly of terminal complexes, the influence of membrane factors will also be studied. In particular, the role of terminal protein interaction with membrane surface proteins will be addressed using photosensitive crosslinking reagents to detect associated proteins. The relevance of these associations to insertion of the terminal complex will then be studied.