STRUCTURE AND FUNCTION OF TERMINAL COMPLEMENT PROTEINS

描述：该项目的重点是分子方面的 人C5 b-9的组装、功能和调节， 补体蛋白C5 b、C6、C7、C8和C9的复合物。 重点 将研究人类C8的结构和功能 （HuC 8），以了解其生物学意义， 其特有 结构，标识符 涉及到发生在 在C5 b-9组装过程中，确定富含半胱氨酸的模块在 调解这些相互作用，确定一个结构基础， C5 b-9调节蛋白对C8的种选择性识别 CD 59，深入了解遗传性C8缺陷，并促进 设计可能具有治疗价值的C8类似物， 具体目标1是映射HuC 8中涉及的结构域， 在C5 b- 7和C8-β、C8-β和C8-α、C8- alpha和C8-gamma，C8-alpha和C9。 HuC 8-α和C8-α的片段 将测试重组DNA表达产生的β的 抑制这些相互作用的能力。 具体目标2是确定 HuC 8-γ的三维结构， 以表征其疏水配体结合特性， α-2u-球蛋白（脂质运载蛋白）家族的特征， 它所属的蛋白质。具体目标3侧重于机制 通过其保护人细胞免受人C5 b-9的损伤，即， 同源限制机制 细胞裂解。 结构 HuC 8-alpha中的决定簇，其在依赖于物种的 将通过HuCD 59的方式进行定位和精细定位。 片段 将比较人和兔C8-α以鉴定那些 以物种依赖性方式与HuCD 59相互作用。 是为了确定兔子C8缺乏的基础。 是什么 最初被认为是C8-α/γ缺乏症，现在看来， C8-alpha/gamma和C8-beta的联合缺乏， 报告在人类。 实验将检验氨基的重要性 已经在兔C8 D-α中检测到的氨基酸取代，并搜索 C8 D-β中的其他取代。 这些实验可以 鉴定C8亚基相互作用关键残基并提供 深入了解HuC 8-alpha/gamma缺乏症的缺陷。 具体目标 5是确定HuC 8-α和C8-α之间的基因间距离， 使用外显子特异性探针和PFGE的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.