Role of USP Ligand Binding Pocket During Development

USP 配体结合袋在开发过程中的作用

• 批准号: 7270045
• 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
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-08-01 至 2009-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7270045
• 关键词: Adult; Aedes; Binding; Biochemical; Biological; Biological Assay; Biological Metamorphosis; Culicidae; Cultured Cells; Development; Dominant-Negative Mutation; Drosophila genus; Ecdysone; Endocrinology; Funding; Future; Hand; Hormonal; Insecta; Juvenile Hormones; Ligand Binding; Ligand Binding Domain; Ligands; Molting; Nuclear Receptors; Outcome; Proteins; RXR; Role; Screening procedure; Signal Transduction; Study Section; Surface; Testing; Transgenic Organisms; United States National Institutes of Health; base; fly; in vivo; insight; mutant

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（由申请人提供）：昆虫内分泌学中一个尚未解决的主要问题是核受体超气门（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伴侣。我们以前的NIH资助的蛋白质生化研究强调了许多点突变体的果蝇USP的配体结合域的筛选。具体目的是确定最佳突变USP，用于2种转基因果蝇试验的体内USP配体结合功能的未来检测。我们的研究得到了其他人最近研究的支持，在培养细胞试验中成功地证明了果蝇（苍蝇）和伊蚊（蚊子）的USP可以抑制苍蝇和蚊子中天然保幼激素（JH）环氧法尼酸甲酯的转录信号。研究还表明，这种细胞内JH信号传导需要USP上的表面区域，其对应于脊椎动物对应物RXR的配体活化的共活化剂结合表面。使用培养的细胞功能测定，我们成功地制备了USP配体结合口袋和共激活剂结合表面的点突变体，其作为显性阴性物阻断培养细胞中的USP依赖性JH信号传导。在此基础上，最近NIH研究科专家组的总结指出，我们下一步的建议是将蛋白质生化研究向前推进 通过使用“已经掌握的突变体，而不是寻找更多的突变体”，现在将研究导向“直接的生物学背景”。因此，在这个新的提议中，我们将这些果蝇USP突变体（和相应的伊蚊USP突变体）用于转基因果蝇试验，以提出以下问题：1）USP配体结合口袋是否是JH/蜕皮素驱动的幼虫到幼虫蜕皮中USP功能所需的？2)USP配体结合口袋对蛹-成虫变态有影响吗？这些体内研究是下一个合乎逻辑的步骤，建立在蛋白质生物化学和培养细胞测定的基础上，是昆虫内分泌学对任何USP的配体结合功能的第一个直接体内测试。这一结果将对昆虫内分泌学产生广泛的影响，从而能够对伊蚊USP进行特定的测试，其结果将对技术上更困难的蚊子的激素信号产生新的见解。