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Role of USP Ligand Binding Pocket During Development

USP 配体结合袋在开发过程中的作用

基本信息

批准号：
7479374
负责人：
GRACE M JONES
金额：
$ 28.48万
依托单位：
UNIVERSITY OF KENTUCKY
依托单位国家：
美国
项目类别：
财政年份：
2005
资助国家：
美国
起止时间：
2005-08-01 至 2011-07-31
项目状态：
已结题

项目摘要

DESCRIPTION (provided by applicant): A major unresolved question in insect endocrinology is whether the nuclear receptor ultraspiracle (USP) has an active, ligand-binding function or, alternatively, is a non-liganded, even passive, heterodimer partner. Our previous NIH-funded protein biochemical studies emphasized screening of many point mutants to the ligand-binding domain of the Drosophila USP. The specific aims were to identify optimal mutant USPs for future testing of an in vivo USP ligand binding function of 2 transgenic fly assays. Our studies, supported by recent studies by others, were successful in demonstrating in cultured cell assays that the USP of Drosophila (flies) and Aedes (mosquitoes) can transduce transcriptional signaling by methyl epoxyfarnesoate, a natural juvenile hormone (JH) in flies and mosquitoes. The studies also show that this intracellular JH signaling requires a surface region on USP corresponding to a ligand-activated co-activator binding surface of the vertebrate counterpart, RXR. Using the cultured cell functional assay, we were successful in preparing point mutants to the USP ligand-binding pocket and co-activator binding surface that act as dominant negatives to block USP-dependent JH signaling in the cultured cells. On this basis, the recent NIH study section panel summary directed that our next proposal move past the protein biochemical studies by using "the mutants already in hand rather than looking for more mutants" and to now orient the studies "into a direct biological context". Therefore, in this new proposal, we take these Drosophila USP mutants (and corresponding Aedes USP mutants) will be used in the transgenic fly assays in order to ask the following questions: 1) Is the USP ligand-binding pocket needed for USP function in JH/ecdysone-driven larval-to-larval molting? 2) Does the USP ligand-binding pocket transduce JH effects on pupal-adult metamorphosis? These in vivo studies are the next logical step, building upon protein biochemical and cultured-cell assays, and are insect endocirinology's first direct in vivo tests of a ligand-binding function for any USP. The outcome will have broad implications for insect endocrinology, enabling specific tests on the Aedes USP, the results of which will yield new insights on hormonal signaling in the technically more difficult mosquito.

描述(申请人提供)：昆虫内分泌学中一个尚未解决的主要问题是，核受体超阿司匹林(USP)是否具有主动的配体结合功能，或者是非配体的，甚至是被动的异二聚体伙伴。我们以前的NIH资助的蛋白质生化研究重点是筛选果蝇USP的配体结合结构域的许多点突变。其具体目的是为未来2种转基因果蝇体内USP配体结合功能的测试寻找最佳的突变体USP。我们的研究在最近其他研究的支持下，成功地在培养细胞分析中证明了果蝇和伊蚊的USP可以通过环氧法尼苏酸甲酯(一种苍蝇和蚊子的天然保幼激素(JH))转导转录信号。研究还表明，这种细胞内的JH信号需要在USP上有一个与脊椎动物RXR的配体激活的共激活子结合表面相对应的表面区。利用培养的细胞功能分析，我们成功地在USP配体结合口袋和共激活子结合表面制备了点突变，作为显性负性阻断培养细胞中USP依赖的JH信号。在此基础上，最近的美国国立卫生研究院研究小组总结指出，我们的下一个提案将超越蛋白质生物化学研究。通过使用“已经在手中的突变体，而不是寻找更多的突变体”，现在将研究定向到“直接的生物学背景”。因此，在这个新的建议中，我们将这些果蝇USP突变体(以及相应的伊蚊USP突变体)用于转基因苍蝇的检测，以提出以下问题：1)USP在JH/蜕皮酮驱动的幼虫到幼虫蜕皮中发挥作用是否需要USP配体结合口袋？2)USP配体结合口袋是否转导JH对幼虫-成虫变态的影响？这些体内研究是下一个合乎逻辑的步骤，建立在蛋白质生化和培养细胞分析的基础上，并且是昆虫内环学对任何USP的配体结合功能的第一次直接体内测试。这一结果将对昆虫内分泌学产生广泛的影响，使对伊蚊USP的特定测试成为可能，其结果将对技术上更困难的蚊子的荷尔蒙信号产生新的见解。