REGULATION OF C1 INHIBITOR SYNTHESIS

C1 抑制剂合成的调控

• 批准号: 6765260
• 负责人: ALVIN E DAVIS
• 金额: $ 31.57万
• 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 2006-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6765260
• 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（改编自研究者摘要）：遗传性血管性水肿 在C1抑制剂（C1 INH）缺乏的杂合子个体中发生。的 单个等位基因的产物不足以控制 通常由C1 INH调节的蛋白水解系统。合理的治疗方法 就是增强这个基因的抑制剂合成。这项建议会 检查尚未研究的C1 INH合成调节的三个方面 广泛存在于任何基因中，并且仍然知之甚少。具体目标1将 检查C1 INH的聚嘌呤-聚嘧啶（Pu-Py）片段的作用 启动子我们将检验这一假设，即增强转录介导的 该区域由转录因子与特异性转录因子相互作用产生。 Pu-Py片段内的序列，而不是通过H-DNA（三螺旋铰链） 阵初步电泳迁移率变动分析和超变动 实验已经证明HNF-1 α（肝细胞核 除此之外，还有几个尚未确定的其他地点， 结合到含有Pu-Py区域内位点的核蛋白 顺序进一步的研究将鉴定和表征这些蛋白质， 它们的功能。其他研究表明Pu-Py区域之间存在协同性， 和HNF-1 α位点。我们将检验这种协同性 结果是转录因子之间的相互作用， 区域通过免疫共沉淀和通过直接分离。dna酶 将进行超敏反应实验，以支持 假设H-DNA的形成发生在体内， 分析使用突变启动子构建体的转录诱导，所述突变启动子构建体 不能形成H-DNA。最后，核小体重建实验 将检验三链体的形成创造了核小体屏障的假设 在复制过程中。 具体目标2将检查磷酸酶在下调细胞凋亡中的作用。 干扰素（IFN）-γ介导的肝细胞中C1 INH诱导比较 与蛋白体降解或STAT 1结合的作用，通过特异性 抑制蛋白初步数据表明，磷酸酶发挥主要作用， 在这一下调中发挥作用。这一假设尚未在肝细胞中得到验证， 它们是大多数血浆蛋白的主要来源，其中许多是 IFN-应答。具体目标3将检验雌激素抑制 C1 INH转录。此外，我们假设， 雄激素是雌激素受体表达下调的结果， 逆转雌激素的作用这里提出的研究将有助于 对C1 INH本身的调节和激素的作用的了解， 核磷酸酶和Pu-Py序列对基因调控的影响。