REGULATION OF C1 INHIBITOR SYNTHESIS

C1 抑制剂合成的调控

• 批准号: 6637255
• 负责人: ALVIN E DAVIS
• 金额: $ 30.47万
• 依托单位: IMMUNE DISEASE INSTITUTE, INC.
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-02-01 至 2005-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6637255
• 关键词: DNA footprinting; androgens; biosynthesis; complement inhibitors; gel mobility shift assay; genetic promoter element; genetic regulation; glucocorticoids; human subject; interferon gamma; nucleic acid sequence; reporter genes; site directed mutagenesis; steroid hormone receptor; transcription factor

DESCRIPTION (adapted from investigator's abstract): Hereditary angioedema develops in individuals heterozygous for C1 inhibitor (C1INH) deficiency. The product of a single allele is insufficient to control activation of the proteolytic systems normally regulated by C1INH. A logical approach to therapy is to enhance inhibitor synthesis from this single gene. This proposal will examine three aspects of C1INH synthesis regulation that have not been studied extensively in any gene and that remain poorly understood. Specific Aim 1 will examine the role of the polypurine-polypyrimidine (Pu-Py) segment of the C1INH promoter. We will test the hypothesis that enhanced transcription mediated by this region results from interaction of transcription factors with specific sequences within the Pu-Py segment rather than via H-DNA (triple helix hinged) formation. Preliminary electrophoretic mobility shift assays and supershift experiments have demonstrated interaction of HNF-1alpha (hepatocyte nuclear factor) with one site in addition to several other, as yet unidentified, nuclear proteins that bind to sites within the region containing the Pu-Py sequence. Further studies will identify and characterize these proteins and their function. Other studies suggest cooperativity between the Pu-Py region and the HNF-1alpha site. We will test the hypothesis that this cooperativity results from interaction between transcription factors that bind to these regions by co-immunoprecipitation and by direct isolation. DNAse hypersensitivity experiments will be performed to provide support for the hypothesis that H-DNA formation takes place in vivo, as will experiments to analyze induction of transcription using mutated promoter constructs that are incapable of H-DNA formation. Lastly, nucleosomal reconstitution experiments will test the hypothesis that triplex formation creates a nucleosomal barrier during replication. Specific Aim 2 will examine the role of phosphatases in down-regulating interferon (IFN)-gamma-mediated induction of C1INH in hepatocytes in comparison with the role of proteosome degradation or binding of STAT1 by specific inhibitory proteins. Preliminary data suggest that phosphatases play a major role in this down-regulation. This hypothesis is unexamined in hepatocytes, which are the primary source of most plasma proteins, many of which are IFN-responsive. Specific Aim 3 will test the hypothesis that estrogens suppress C1INH transcription. Furthermore, we hypothesize that the therapeutic effect of androgens is a result of down-regulation of estrogen receptor expression, which reverses the estrogenic effect. The studies proposed here will contribute both to knowledge of the regulation of C1INH itself and to the role of hormones, nuclear phosphatases and Pu-Py sequences on gene regulation in general.

描述(摘自研究人员摘要)：遗传性血管性水肿 在C1INH缺乏的杂合子个体中发展。这个 单个等位基因的产物不足以控制基因的激活 蛋白分解系统通常由C1INH调节。治疗的合乎逻辑的方法 是为了加强从这个单一基因合成抑制物。这项提议将 考察尚未研究过的C1INH合成调控的三个方面 在任何基因中都广泛存在，但人们对此仍知之甚少。具体目标1将 研究C1INH的多嘌呤-多嘧啶(Pu-Py)片段的作用 推动者。我们将检验这样一种假设，即通过 这个区域是转录因子与特定基因相互作用的结果。 Pu-Py片段内的序列而不是通过H-DNA(三螺旋铰链) 队形。初步的电泳迁移率变化分析和超迁移 实验证明了HNF-1α(肝细胞核)的相互作用 因素)，除了一个地点外，还有其他几个地点， 与含有Pu-Py的区域内的位置结合的核蛋白 序列。进一步的研究将鉴定和鉴定这些蛋白质和 它们的功能。其他研究表明，Pu-Py区域之间存在协同作用 和HNF-1α位点。我们将检验这样一个假设，即这种合作性 结合这些转录因子的转录因子之间相互作用的结果 免疫共沉淀法和直接分离法。DNA酶 将进行过敏性实验，为 假设H-DNA在体内形成，实验也将如此 使用突变的启动子结构分析转录的诱导 不能形成H-DNA。最后，核小体重组实验。 将检验三链形成会产生核小体屏障的假说 在复制期间。 特定目标2将研究磷酸酶在下调中的作用 干扰素-γ诱导肝细胞C1INH的比较 与蛋白酶体降解或通过特异性结合STAT1的作用 抑制蛋白。初步数据表明，磷酸酶起着主要作用 在这一下调监管中扮演的角色。这一假说在肝细胞中未经检验， 它们是大多数血浆蛋白的主要来源，其中许多是 干扰素反应。《特指目标3》将检验雌激素抑制 C1INH转录。此外，我们还假设阿司匹林的疗效 雄激素是雌激素受体表达下调的结果， 逆转雌激素的作用。这里提出的研究将对 对C1INH本身的调节和激素的作用的了解， 核磷酸酶和Pu-Py序列对基因调控的一般作用。