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.

目的：蜕皮激素和保幼激素(JH)是调节昆虫生活史中各种发育事件的主要激素。蜕皮激素的多种生物学效应是由蜕皮激素受体(ECR)及其异二聚体超阿司匹林(USP)介导的。JH作用的分子机制还不是很清楚。JH和蜕皮类固醇的生物学效应表明这两种激素之间存在相互作用。利用JH-和20-羟基蜕皮激素(20E)反应性JH酯酶(JHE)基因启动子区域的JH反应元件(JHRE)和蜕皮激素受体(ECR)缺陷的黑腹果蝇细胞系(L57)建立了一个健壮的系统。在L57细胞中，JHRE控制下的报告基因被JH诱导，20E以剂量和时间依赖的方式抑制JH诱导的表达。从L57细胞中分离的核蛋白与JHRE特异结合，这种结合可被20E预处理所阻止。这种20E抑制JH诱导的基因表达是通过ECR介导的。初步研究表明，ECR不直接与含有JHRE的启动子结合。20E抑制JH作用需要的是ECR的配体结合而不是DNA结合。这些结果表明，ECR通过一种“非经典”的作用模式抑制JH诱导的基因表达，即不直接与蜕皮激素反应元件结合。这是第一个提出ECR的非经典作用量的系统的例子。该系统将用于研究ECR的非经典作用机制及其与JH信号转导级联的串扰。这项建议的两个具体目标是：(1)研究20E抑制JH反应的机制；(2)鉴定和鉴定在20E和JH之间的串扰中重要的蛋白质。突变以选择黑腹果蝇ECR配体结合域中的氨基酸将被用来验证ECR的非经典作用。将利用RNA干扰(RNAi)介导的USP、果蝇激素受体38和78以及Seven-up基因的沉默来研究它们在20E抑制JH反应中的作用。酵母双杂交试验将用于鉴定与ECR相互作用的蛋白质。与ECR相互作用的蛋白质将通过(I)RNAi介导的沉默其在L57细胞中的表达，(Ii)下拉和双杂交试验，(Iii)在黑腹果蝇组织中的RNA表达的研究，以及(Iv)对黑腹果蝇突变体中编码已识别蛋白质的基因的JH和20E反应的分析来表征。