REGULATION OF C1 INHIBITOR SYNTHESIS

C1 抑制剂合成的调控

• 批准号: 6387694
• 负责人: ALVIN E DAVIS
• 金额: $ 30.96万
• 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/6387694
• 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（改编自研究者的摘要）：遗传性血管性水肿 发生在 C1 抑制剂 (C1INH) 缺乏杂合子个体中。这 单个等位基因的产物不足以控制 蛋白水解系统通常受 C1INH 调节。合理的治疗方法 是增强这个单一基因的抑制剂合成。该提案将 检查尚未研究的 C1INH 合成调控的三个方面 广泛存在于任何基因中，但人们对此仍知之甚少。具体目标 1 将 检查 C1INH 的聚嘌呤-聚嘧啶 (Pu-Py) 片段的作用 发起人。我们将检验以下假设：转录增强是由 该区域是转录因子与特定的相互作用的结果 Pu-Py 片段内的序列，而不是通过 H-DNA（三螺旋铰接） 形成。初步电泳迁移率变动分析和超迁移 实验证明了 HNF-1α（肝细胞核）的相互作用 因素）与一个站点以及其他几个尚未确定的站点， 与包含 Pu-Py 的区域内的位点结合的核蛋白 顺序。进一步的研究将鉴定和表征这些蛋白质 他们的功能。其他研究表明 Pu-Py 区域之间存在协同作用 和 HNF-1α 位点。我们将检验这种协作性的假设 与这些转录因子结合的转录因子之间相互作用的结果 通过免疫共沉淀和直接分离的区域。脱氧核糖核酸酶 将进行超敏反应实验以提供支持 H-DNA 形成发生在体内的假设，以及实验 使用突变的启动子构建体分析转录的诱导 不能形成H-DNA。最后，核小体重建实验 将检验三链体形成产生核小体屏障的假设 复制期间。 具体目标 2 将检查磷酸酶在下调中的作用 干扰素 (IFN)-γ 介导的肝细胞 C1INH 诱导比较 与蛋白质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发挥着主要作用 在这种下调中发挥作用。这一假设尚未在肝细胞中得到检验， 它们是大多数血浆蛋白的主要来源，其中许多是 IFN 反应性。具体目标 3 将检验雌激素抑制的假设 C1INH 转录。此外，我们假设治疗效果 雄激素是雌激素受体表达下调的结果， 逆转雌激素作用。这里提出的研究将有助于 了解 C1INH 本身的调节以及激素的作用， 核磷酸酶和 Pu-Py 序列对基因调控的一般影响。