COLLABORATIVE PROJECT: Receptors for Juvenile Hormones and JH Mimics

这项拟议的研究是对结合和降解保幼激素(JH)和JH模拟物(JHM)的蛋白质的结构、动力学和生理学的跨学科合作的继续。这项合作研究的最初目标是开发化学和生化技术，以接近保幼激素(JH)接收和分解代谢的分子生物学。这些初步目标已经达到，在这次更新请求中，提出了进一步的目标：第一，通过(A)放射性配体的合成，(B)JHBP/RP和JHE/EH纯化和测序，研究Manduca幼虫表皮细胞胞浆和细胞核中JH和JHM结合蛋白(JHBP和RP)的结构，以及JH酯酶(JHE)和环氧化物水解酶(JHEH)的结构。(C)活性部位标记和测序；(D)抗体生产；(E)基因分离和测序；(F)蛋白质生产过剩；第二，研究配体和大分子的动力学，包括(A)JHBP/RP、JHE/EH的时间和位置，(B)激素和蛋白质在细胞间的移动，以及(C)蛋白质-配体、蛋白质-蛋白质和蛋白质-DNA的相互作用。结构和动力学目标的关键将是在家蚕中开发杆状病毒表达系统，以解决蛋白质过度生产的问题。第三个目标是通过表征(A)JHBP/RP和JHE/EH在生命周期中的出现，以及(B)JHBP/RP和JHE/EH在昆虫中的比较生物化学，从配体识别和蛋白质相似性方面，将结构和动力学与生理学联系起来。保幼激素受体蛋白(JH)及其类似物(JHM)的特性及其降解途径的了解是理解昆虫激素调控基因表达的分子基础的关键。这项研究是一项跨学科的合作，结合了昆虫分子生物学和基因表达的生理表现(L.M.Riddiford)，JH代谢的生化和分子研究(B.D.Hammock)，以及高比活性标记JHS和JHMS的合成和使用(G.D.Prestwich)。这项提案是根据NSF在生命过程化学中的倡议提交的。

The proposed research is a continuation of an interdisciplinary collaboration on the structure, dynamics, and physiology of proteins which bind and degrade juvenile hormone (JH) and JH mimics (JHM). The original goals of this collaborative research were to develop chemical and biochemical techniques to approach the molecular biology of juvenile hormone (JH) reception and catabolism. These initial objectives have been acheived, and in this renewal request, further goals are proposed: First, to study the structure of the JH and JHM binding proteins (JHBP an RP) in Manduca larval epidermal cell cytosol and nuclei, and of JH esterases (JHE) and epoxide hydrolases (JHEH) through (a) synthesis of radioligands, (b) JHBP/RP and JHE/EH purification and sequencing, (c) active site labeling and sequencing, (d) antibody production, (e) gene isolation and sequencing, and (f) overproduction of proteins; second, to examine the dynamics of the ligands and macromolecules, including (a) timing and location of JHBP/RP, JHE/EH, (b) movement of hormones and proteins between cell compartments, and (c) protein-ligand, protein-protein, and protein-DNA interactions. A key to the structure and dynamics goals will be the development of a baculovirus expression system in Bombyx for protein overproduction. The third goal is to correlate structure and dynamics with physiology by characterizing, (a) the appearance of JHBP/RP and JHE/EH in the life cycle, and (b) comparative biochemistry of JHBP/RP and JHE/EH in insects, with respect to ligand recognition and protein similarities. The characterization of receptor proteins for juvenile hormone (JH) and JH mimics (JHM) and knowledge of the pathways of their degradation are major keys to understanding the molecular basis for hormonal regulation of gene expression in insects. This research is an interdisciplinary collaboration which combines expertise in molecular biology and physiological manifestations of gene expression in insects (L. M. Riddiford), biochemical and molecular studies of JH metabolism (B. D. Hammock), and synthesis and use of high specific activity labeled JHs and JHMs (G. D. Prestwich). This proposal was submitted under the NSF initiative in the Chemistry of Life Processes.