AH RECEPTOR ANATOMY--IMPLICATIONS FOR DIOXIN TOXICITY

AH 受体解剖——二恶英毒性的影响

• 批准号: 2377914
• 负责人: Cornelis Johan Elferink
• 金额: $ 9.83万
• 依托单位: WAYNE STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-03-01 至 2001-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2377914
• 关键词: DNA binding protein; aromatic hydrocarbon receptor; carbopolycyclic compound; cell cell interaction; chimeric proteins; complementary DNA; dioxins; environmental toxicology; gel mobility shift assay; immunocytochemistry; immunoprecipitation; laboratory rabbit; laboratory rat; molecular cloning; oligonucleotides; polymerase chain reaction; protein sequence; protein structure function; receptor binding; receptor expression; toxicant interaction; toxin metabolism; western blottings

Inconclusive epidemiological studies on the risks of environmental contaminants, exemplified by the human health concerns over 2,3,7,8- tetrachlorodibenzo-p-dioxin (TCDD, dioxin), emphasizes the need for a mechanistic approach to these toxicological issues. Most, if not all the biological effects of TCDD are mediated through the aromatic hydrocarbon (Ah) receptor. This research proposal centers on the premise that a mechanistic understanding of TCDD toxicity, is contingent upon identifying and characterizing all the components involved in Ah receptor function. Following binding by TCDD, the Ah receptor binds to specific DNA enhancer elements to regulate gene expression. Target genes include those encoding drug metabolizing enzymes and growth factors important in normal cell growth and differentiation. The Ah receptor-DNA interaction involves the Ah receptor nuclear translocator (Arnt) protein, a DNA-binding partner. We previously identified a 110 kDa as part of the Ah receptor DNA-binding complex. Here, our preliminary research identifies a distinct 110 kDa DNA- binding protein that copurifies with both the rat Ah receptor and Arnt protein. Peptide sequencing of 110 kDa protein fragments reveals no homology to known proteins. Our data suggests that the 110 kDa protein represents an additional component of the Ah receptor DNA-binding complex. Functional, genetic and biochemical evidence from others provides further support for our hypothesis that Ah receptor DNA binding and function involves association with factors other than the Arnt protein. Therefore, this proposal seeks to define the role of the 110 kDa protein in Ah receptor DNA-binding and function, in the context of our long range objective of understanding TCDD-induced intracellular signaling. We propose four specific aims to test the hypothesis: 1. cDNA cloning the rat 110 kDa protein. cDNA clones will be isolated as a first step towards learning the function of the 110 kDa protein. Amino acid microsequencing on several peptide fragments from the purified 110 kDa protein permits design of degenerate oligonucleotides intended for use in either of two cDNA cloning strategies. 2. The isolation of cDNA clones for the human homolog of the rat 110 kDa protein. In order to study the role of the 110 kDa protein in human Ah receptor function, cDNA clones for the human protein will be isolated using the rat clones as probes, and used in expression studies. 3. Studies using the yeast two-hybrid system. The two-hybrid system represents a third alternative for isolating 110 kDa protein clones, that is distinct from the two strategies using the degenerate oligonucleotides. The two-hybrid system also permits an in vivo examination of functionally important protein-protein interactions between the 110 kDa protein, Ah receptor and Arnt protein. 4. Expression studies to examine the role of the 110 kDa protein in Ah receptor DNA binding and function. Studies will focus on identifying the important protein domains i the 110 kDa protein and their impact on Ah receptor and Arnt protein DNA binding and function.

关于环境风险的非结论性流行病学研究 污染物，以人类健康问题为例 2,3,7,8- 四氯二苯并-对-二恶英（TCDD，二恶英），强调需要 这些毒理学问题的机械方法。大多数（如果不是全部） TCDD 的生物效应是通过芳香烃介导的 （啊）受体。本研究提案的前提是 对 TCDD 毒性的机制理解取决于识别 并表征参与 Ah 受体功能的所有组件。 与 TCDD 结合后，Ah 受体与特定的 DNA 增强子结合 调节基因表达的元件。靶基因包括那些编码 对正常细胞很重要的药物代谢酶和生长因子 生长和分化。 Ah 受体-DNA 相互作用涉及 Ah 受体核转位蛋白 (Arnt) 蛋白，一种 DNA 结合伴侣。我们 先前鉴定出 110 kDa 作为 Ah 受体 DNA 结合的一部分 复杂的。在这里，我们的初步研究确定了一个独特的 110 kDa DNA- 与大鼠 Ah 受体和 Arnt 共纯化的结合蛋白 蛋白质。 110 kDa 蛋白质片段的肽测序表明没有 与已知蛋白质的同源性。我们的数据表明 110 kDa 蛋白质 代表 Ah 受体 DNA 结合复合物的附加成分。 来自其他人的功能、遗传和生化证据进一步提供了 支持我们的假设，即 Ah 受体 DNA 结合和功能 涉及与 Arnt 蛋白以外的因素的关联。所以， 该提案旨在定义 110 kDa 蛋白质在 Ah 中的作用 受体 DNA 结合和功能，在我们的长距离范围内 目的是了解 TCDD 诱导的细胞内信号传导。我们 提出四个具体目标来检验假设： 1. cDNA 克隆大鼠 110 kDa 蛋白。 cDNA 克隆将被分离为 了解 110 kDa 蛋白质功能的第一步。氨基 对纯化的 110 中的几个肽片段进行酸微测序 kDa 蛋白允许设计要使用的简并寡核苷酸 两种 cDNA 克隆策略中的任一种。 2. 大鼠 110 kDa 人类同源物 cDNA 克隆的分离 蛋白质。为了研究110 kDa蛋白在人体Ah中的作用 受体功能，将分离人类蛋白质的 cDNA 克隆 使用大鼠克隆作为探针，并用于表达研究。 3.利用酵母二杂交系统进行研究。二元混合系统 代表分离 110 kDa 蛋白质克隆的第三种选择，即 与使用简并寡核苷酸的两种策略不同。 双杂交系统还允许对功能进行体内检查 110 kDa 蛋白质 Ah 之间重要的蛋白质-蛋白质相互作用 受体和 Arnt 蛋白。 4. 表达研究以检查 110 kDa 蛋白在 Ah 中的作用 受体 DNA 结合和功能。 研究将侧重于确定 重要的蛋白质结构域 i 110 kDa 蛋白质及其对 Ah 的影响 受体与Arnt蛋白DNA结合并发挥作用。