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（根据调查员的摘要改编）：遗传性血管性水肿 C1抑制剂（C1INH）缺乏的个体发育。这 单个等位基因的产物不足以控制激活 蛋白水解系统通常由C1INH调节。逻辑治疗方法 是为了增强该单个基因的抑制剂合成。该提议将 检查尚未研究的C1inh合成调节的三个方面 在任何基因中都广泛理解。具体目标1将 检查C1INH的息肉素二甲酰胺（PU-PY）段的作用 发起人。我们将检验以下假设，即增强了由 该区域是由于转录因子与特定的相互作用而引起的 PU-PY段内而不是通过H-DNA（三重螺旋铰链）内的序列 形成。初步的电泳移动性转移测定法和超速移动 实验证明了HNF-1Alpha（肝细胞核）的相互作用 因素）除了一个尚未确定的其他几个站点外，还有一个地点 与包含PU-PY区域内的位点结合的核蛋白 顺序。进一步的研究将识别和表征这些蛋白质，并 他们的功能。其他研究表明PU-PY区域之间的合作 和HNF-1Alpha网站。我们将测试这种合作性的假设 与这些结合的转录因子之间的相互作用的结果 通过共免疫沉淀和直接隔离区域。 DNase 将进行超敏反应实验，以提供支持 假设H-DNA形成发生在体内，实验也是如此 使用突变的启动子构建体分析转录的诱导 无法形成H-DNA。最后，核小体重构实验 将检验以下假设，即三层形成会产生核小体屏障 在复制过程中。 特定目标2将检查磷酸酶在下调中的作用 相比 蛋白体降解或STAT1结合的作用 抑制蛋白。初步数据表明，磷酸酶发挥作用 在这种下调中的作用。该假设在肝细胞中未经检查， 这是大多数血浆蛋白的主要来源，其中许多是 IFN响应。特定目标3将检验雌激素抑制的假设 C1inh转录。此外，我们假设 雄激素是雌激素受体表达下调的结果， 逆转雌激素作用。这里提出的研究将兼具 了解C1inh本身的调节以及激素的作用， 一般而言，基因调节的核磷酸酶和PU-PY序列。