COLLABORATIVE PROJECT: Receptors for Juvenile Hormones and JH Mimics

拟议的研究是对结合和降解少年激素（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延续。这项合作研究的最初目标是发展化学和生化技术，以接近少年激素（JH）的接受和分解代谢的分子生物学。这些最初的目标已经实现，在这项延长期限的请求中，提出了进一步的目标：首先，通过(a)放射性配体的合成，(b) JHBP/RP和JHE/EH的纯化和测序，(c)活性位点的标记和测序，(d)抗体的产生，(e)基因的分离和测序，研究Manduca幼虫表皮细胞质和细胞核中JH和JHM结合蛋白（JHBP和RP）以及JH酯酶（JHE）和环氧化物水解酶（JHEH）的结构。(f)蛋白质生产过剩；其次，检查配体和大分子的动力学，包括(a) JHBP/RP， JHE/EH的时间和位置，(b)细胞间室之间激素和蛋白质的运动，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实现蛋白质的过量生产。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在昆虫体内的比较生物化学，将结构和动力学与生理学联系起来。关于配体识别和蛋白质相似性。研究昆虫幼体激素（JH）和幼体激素模拟物（JHM）的受体蛋白及其降解途径是了解昆虫激素调控基因表达的分子基础的关键。这项研究是一个跨学科的合作，结合了分子生物学和昆虫基因表达的生理表现的专业知识（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jhm的合成和使用（G. D. Prestwich）。该提案是在美国国家科学基金会生命过程化学倡议下提交的。

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.