20-Hydroxyecdysone Suppression of Juvenile Hormone Response

20-羟基蜕皮酮抑制保幼激素反应

• 批准号: 0421856
• 负责人: Subba Palli
• 金额: $ 47.23万
• 依托单位: University of Kentucky Research Foundation
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-08-01 至 2014-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0421856&HistoricalAwards=false
• 关键词: 20; Hydroxyecdysone; Suppression; Juvenile; Hormone

Title: 20-hydroxyecdysone suppression of juvenile hormone actionPI: Subba Reddy PalliAbstract:Ecdysteroids and juvenile hormone (JH) are major hormones that regulate various developmental events during an insect's life cycle. The diverse biological effects of ecdysteroids are mediated by ecdysone receptor (EcR) and its heterodimeric partner, ultraspiracle (USP). The molecular mechanisms of JH action are not well understood. The biological effects of JH and ecdysteroids suggest a cross talk between these two hormones. JH response elements (JHRE) identified in the promoter region of JH- and 20-hydroxyecdysone (20E)-responsive JH esterase (jhe) gene and ecdysone receptor (EcR) deficient Drosophila melanogaster cell line (L57) were used to develop a robust system. In L57 cells, a reporter gene placed under the control of JHRE is induced by JH, and the JH-induced expression is suppressed by 20E in a dose- and time-dependent manner. The nuclear proteins isolated from L57 cells specifically bound to JHRE and this binding was prevented by pretreatment of nuclear proteins with 20E. This 20E suppression of JH-induced gene expression is mediated through EcR. Preliminary studies showed that EcR does not directly bind to JHRE containing promoter. Ligand binding but not DNA binding of EcR is required for 20E suppression of JH action. These results suggest that the EcR suppresses JH-induced gene expression though a "nonclassical" mode of action, i.e., without binding to ecdysone response elements directly. This is the first example of a system where a nonclassical action for EcR is suggested. This system will be used to study the mechanisms involved in the nonclassical action of EcR and its cross talk with the components of JH signal transduction cascade. The two specific objectives of this proposal are (1) to study the mechanism for 20E suppression of JH response and (2) to identify and characterize proteins that are important in cross talk between 20E and JH. Mutations to select amino acids in the ligand binding domain of D. melanogaster EcR will be used to verify nonclassical action of EcR. RNA interference (RNAi)-mediated silencing of usp, Drosophila hormone receptors 38 and 78, and Seven-up genes will be employed to study their role in 20E suppression of JH response. A yeast two-hybrid assay will be used to identify proteins that interact with EcR. The EcR-interacting proteins will be characterized through (i) RNAi-mediated silencing of their expression in L57 cells, (ii) pull down and two-hybrid assays, (iii) studies on expression of RNA in D. melanogaster tissues and (iv) analysis of JH and 20E response in D. melanogaster mutants for genes coding for the identified proteins.

标题：20-羟基丁香抑制少年激素动作：Subba reddy palliabstract：ecdys-甾体和幼年激素（JH）是在昆虫生命周期期间调节各种发育事件的主要激素。 ecdy-甾体类固醇的不同生物学作用是由ecdysone受体（ECR）及其异二聚体伴侣Ultraspiracle（USP）介导的。 JH作用的分子机制尚不清楚。 JH和eDysteroid的生物学作用表明，这两种激素之间进行了交叉说话。 JH响应元件（JHRE）在JH-和20-羟基甲虫（20E）的启动子区域（20E） - 反应JH酯酶（JHE）基因（JHE）基因和Ecdysone受体（ECR）缺乏Drosophila melanogaster细胞系（L57）用于发展强大的系统。 在L57细胞中，JH诱导置于JHRE控制下的报告基因，JH诱导的表达以剂量和时间依赖性方式抑制20E。 从L57细胞中分离出的核蛋白特异性与JHRE结合，并通过20E预处理核蛋白来阻止这种结合。 JH诱导的基因表达的20E抑制是通过ECR介导的。 初步研究表明，ECR不直接与含有JHRE的启动子结合。 抑制JH作用需要配体结合，而不是ECR的DNA结合。 这些结果表明，ECR抑制了JH诱导的基因表达，即一种“非经典”的作用方式，即直接与Ecdysone响应元素结合而没有结合。 这是建议对ECR进行非经典作用的系统的第一个示例。 该系统将用于研究ECR非经典作用的机制及其与JH信号转导级联的组成部分进行交流。 该提案的两个具体目标是（1）研究20E抑制JH反应的机制，以及（2）识别和表征在20E和JH之间进行串扰中很重要的蛋白质。 在D. melanogaster ECR的配体结合结构域中选择氨基酸的突变将用于验证ECR的非经典作用。 RNA干扰（RNAi）介导的USP，果蝇受体38和78的沉默以及78个基因将用于研究其在20E抑制JH反应中的作用。 酵母两杂交测定法将用于鉴定与ECR相互作用的蛋白质。 ECR相互作用蛋白将通过（i）RNAi介导的其在L57细胞中的表达沉默，（ii）下拉和两个杂交测定，（iii）关于甲状腺菌组织中RNA表达的研究（III） ）分析用于鉴定蛋白质的基因的黑色素d。melanogaster突变体中的JH和20E反应。