STRUCTURE AND FUNCTION OF TERMINAL COMPLEMENT PROTEINS

描述：这个项目主要关注的是分子方面的 人细胞溶解因子C5b-9的组装、功能及调控 补体蛋白C5b、C6、C7、C8和C9的复合体。重点 将会描述人类C8的结构和功能 (HuC8)作为理解其特殊的生物学意义 结构，确定发生的交互中涉及的域 在C5b-9组装过程中，确定富含半胱氨酸的模块在 调解这些交互作用，确定 C5b-9调节蛋白对C8的物种选择性识别 CD59，深入了解遗传性C8缺陷并促进 可能具有细胞溶解治疗价值的C8类似物的设计 代理。具体目标1是映射HuC8内涉及的域 在C5b-7与C8-β、C8-β与C8-α、C8- 阿尔法和C8-伽马，C8-阿尔法和C9。HuC8-α和C8-的碎片 通过重组DNA表达产生的β将被测试其 抑制这些相互作用的能力。具体目标2是确定 用晶体分析和电子能谱研究HuC8-Gamma的三维结构 以表征其疏水配体结合特性，这些特性是 猪α-2U球蛋白(Lipocalin)家族的特性 它所属的蛋白质。具体目标3侧重于机制 通过它保护人类细胞免受人类C5b-9的破坏，即 细胞裂解的同源限制机制。结构性 HuC8-α中的决定因素在一个物种依赖的 HuCD59的方式将本地化并进行精细映射。片断来自 人类和兔子的C8-α将进行比较，以确定哪些 以依赖物种的方式与HuCD59相互作用。 是为了找出兔体内C8缺乏的基础。什么是 最初被认为是C8-α/伽马缺乏，现在似乎是 类似于C8-α/伽马、C8-β的综合缺乏症 在人类身上也有报道。实验将检验氨基的重要性。 在兔C8D-α中已检测到的酸替代并寻找 C8D-BETA中的其他替代。这样的实验可能 确定对C8亚基相互作用至关重要的残基并提供 洞察HuC8-α/γ缺乏症的缺陷。特定目标 5是确定HuC8-α和C8-α之间的基因间距离。 用外显子特异性探针和PFGE研究β基因座及其定位 分析。

DESCRIPTION: This project focuses on molecular aspects of the assembly, function and regulation of human C5b-9, the cytolytic complex of complement proteins C5b, C6, C7, C8, and C9. Emphasis will be on characterizing the structure and function of human C8 (HuC8) as to understand the biological significance of its peculiar structure, identify domains involved in interactions that occur during C5b-9 assembly, determine the role of cysteine- rich modules in mediating these interactions, identify a structural basis for the species-selective recognition of C8 by the C5b-9-regulatory protein CD59, gain insight into hereditary C8 deficiencies and facilitate the design of C8 analogues that might have therapeutic value as cytolytic agents.Specific Aim 1 is to map domains within HuC8 that are involved in interactions between C5b- 7 and C8-beta, C8-beta and C8-alpha, C8- alpha and C8-gamma, and C8-alpha and C9. Fragments of HuC8-alpha and C8- beta produced by recombinant DNA expression will be tested for their ability to inhibit these interactions. Specific Aim 2 is to determine the 3-D structure of HuC8-gamma through crystallographic analyses and to characterize its hydrophobic ligand binding properties which are characteristic of the alpha-2u-globulin (lipocalin) family of proteins to which it belongs. Specific Aim 3 focuses on the mechanism by which human cells are protected from damage by human C5b-9, i.e. the mechanism of homologous restriction of cell lysis. Structural determinants in HuC8-alpha that are recognized in a species-dependent manner by HuCD59 will be localized and fine-mapped. Fragments from human and rabbit C8-alpha will be compared to identify those that interact with HuCD59 in a species-dependent manner.Specific Aim 4 is to identify the basis of C8 deficiency in the rabbit. What was initially believed to be a C8-alpha/gamma deficiency now appears to be a combined C8-alpha/gamma, C8-beta deficiency analogous to that reported in humans. Experiments will examine the importance of amino acids substitutions already detected in rabbit C8D-alpha and search for additional substitutions in C8D-beta. Such experiments could identify residues critical for C8 subunit interaction and provide insight into the defect in HuC8-alpha/gamma deficiency. Specific Aim 5 is to determine the intergenic distance between HuC8-alpha and C8- beta loci and their orientation using exon-specific probes and PFGE analysis.