20-Hydroxyecdysone Suppression of Juvenile Hormone Response

职务名称：20-羟基蜕皮激素抑制保幼激素作用PI：Subba Reddy Palli摘要：蜕皮类固醇和保幼激素（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）及其异源二聚体伴侣超气门（USP）介导的。 JH作用的分子机制还不清楚。 JH和蜕皮激素的生物学效应表明这两种激素之间存在相互作用。 在JH-和20-羟基蜕皮激素（20 E）-响应性JH酯酶（jhe）基因的启动子区鉴定的JH响应元件（JHRE）和蜕皮激素受体（EcR）缺陷的果蝇细胞系（L57）被用于建立稳健的系统。 在L57细胞中，置于JHRE控制下的报告基因被JH诱导，并且JH诱导的表达以剂量和时间依赖性方式被20 E抑制。 从L57细胞中分离的核蛋白特异性结合JHRE，并且这种结合通过用20 E预处理核蛋白而被阻止。 这种20 E抑制JH诱导的基因表达是通过EcR介导的。 初步研究表明，EcR不直接结合JHRE启动子。 20 E抑制JH的作用需要配体结合而不是DNA结合。 这些结果表明，EcR通过“非经典”作用模式抑制JH诱导的基因表达，即，而不直接结合蜕皮激素应答元件。 这是第一个例子的系统中，一个非经典行动的EcR的建议。 该系统将用于研究EcR的非经典作用机制及其与JH信号转导级联反应组分的相互作用。 本提案的两个具体目标是（1）研究20 E抑制JH反应的机制和（2）鉴定和表征在20 E和JH之间的串扰中重要的蛋白质。 突变以选择D.黑腹果蝇EcR将用于验证EcR的非经典作用。 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20 E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。 将通过（i）RNAi介导的L57细胞表达沉默，（ii）pull down和双杂交测定，（iii）D.黑腹果蝇组织的JH和20 E反应分析。黑腹果蝇突变体中编码所鉴定蛋白质的基因。

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.