Neuropeptide Control of Ecdysone Biosynthesis

蜕皮激素生物合成的神经肽控制

• 批准号: 0130825
• 负责人: Lawrence Gilbert
• 金额: $ 49.21万
• 依托单位: University of North Carolina at Chapel Hill
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-01-15 至 2005-12-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0130825&HistoricalAwards=false
• 关键词: Neuropeptide; Control; Ecdysone; Biosynthesis

In order for insects to grow and undergo metamorphosis, they must shed their restrictive outer skin (exoskeleton). They have used this process very efficiently over hundreds of millions of years in order to grow at times when predators and other endangering environmental factors are less threatening. Many of the structural, physiological, biochemical and molecular events that occur during the molting process are elicited by the principle molting hormone of insects, a polyhydroxylated steroid hormone 20-hydroxyecdysone, which is synthesized in many tissues of the insect from another steroid, ecdysone. Ecdysone is synthesized in special glands in the insect, the prothoracic glands. The stimulus to synthesize this very important molecule is a peptide, prothoracicotropic hormone, secreted by four specialized neurosecretory cells in the insect's brain. About 40 years ago, ecdysone was extracted and characterized from two tons of commercial silkworm, but during the past four decades there has been little progress in elucidating the individual biochemical steps in ecdysone synthesis. Understanding the synthetic steps is important not only to further our basic knowledge of this very important group of animals that intimately affects human welfare, but also because if one is able to characterize the individual steps in the ecdysone biosynthetic pathway, and clone the genes for the enzymes that mediate each of these steps, there is the possibility of introducing this sequence of genes (transfection) into agriculturally important plants. They would then synthesize this steroid hormone, which is a natural deterrent to insect pests. Thus, when the hormone is ingested by an insect consuming such transfected plants, it will molt out of synchrony and will not be viable. Ecdysone is nontoxic to higher organisms, so that this possibility of introducing the necessary genes into host plants is a reasonable goal. Little progress had been made using classical biochemical techniques to elucidate the pathway of ecdysone biosynthesis because the intermediate compounds are in very low quantity and are extremely unstable. Almost all laboratories that have attempted this feat have given up after years of negative results. In collaboration with the O'Connor laboratory at the University of Minnesota, the principal investigator has made critical progress in elucidating the biosynthetic pathway between cholesterol and ecdysone. For these studies, the fruitfly Drosophila melanogaster has been employed because its genetics are better known than any other animal, and its short life cycle makes both the molecular genetics and biochemistry more feasible. This combined use of molecular genetics and biochemical analysis has resulted in the identification, cloning, and functional genomic studies of two enzymes in the ecdysone biosynthetic pathway, and has provided insights into experimental approaches for elucidating the remaining enzymes in that pathway. By cloning and sequencing certain genes (Halloween genes) from Drosophila embryos that have less than the normal amount of ecdysone, the principal investigator and his colleagues learned that mutations in these genes cause lethality in the embryo. Transfection of these genes into a Drosophila cell line and use of biochemical technology has shown that two of these mutants, disembodied and shadow, code for mitochondrial ecdysterol 22- and 2-hydroxylases, respectively. The purpose of continued work over the next several years will be to fully identify these two enzymes through more sophisticated analyses (mass spectrometry) and identify as many of the remaining (5) enzymes (P450 enzymes) in this biosynthetic pathway. Expression of these genes will be examined during Drosophila development, and a search will be conducted for counterparts in other insect model systems, such as the tobacco hornworm. Not only will these experiments result in a complete knowledge of how ecdysone is biosynthesized, but they will also provide the genes for transfection into plants of agricultural importance. In addition, the data should provide a clear understanding of the rate-limiting control mechanisms that modulate ecdysone biosynthesis, and perhaps will permit identification of the specific biosynthetic reactions that are controlled by the brain neuropeptide, prothoracicotropic hormone. This new paradigm of combining molecular genetics with biochemical analysis can be used in the future for studying biosynthetic pathways in a variety of organisms, including pathogens.

为了让昆虫生长并经历变态，它们必须脱落其限制性的外皮（外骨骼）。数亿年来，它们非常有效地利用了这一过程，以便在捕食者和其他危险环境因素威胁较小时生长。蜕皮过程中发生的许多结构、生理、生化和分子事件都是由昆虫的主要蜕皮激素（一种多羟基类固醇激素 20-羟基蜕皮激素）引起的，它是在昆虫的许多组织中由另一种类固醇蜕皮激素合成的。蜕皮激素是在昆虫的特殊腺体——前胸腺中合成的。合成这种非常重要的分子的刺激物是一种肽，即促胸腺激素，由昆虫大脑中的四个专门的神经分泌细胞分泌。大约 40 年前，从两吨商业蚕中提取并表征了蜕皮激素，但在过去的四十年中，在阐明蜕皮激素合成的各个生化步骤方面几乎没有进展。了解合成步骤非常重要，不仅可以加深我们对这一与人类福祉密切相关的非常重要的动物群的基本了解，而且还因为如果能够表征蜕皮激素生物合成途径中的各个步骤，并克隆介导每个步骤的酶的基因，就有可能将这一基因序列（转染）引入具有重要农业意义的植物中。然后他们会合成这种类固醇激素，这是对害虫的天然威慑作用。因此，当激素被食用此类转染植物的昆虫摄入时，它会不同步蜕皮并且无法存活。蜕皮激素对高等生物体是无毒的，因此将必要的基因引入宿主植物的可能性是一个合理的目标。使用经典生化技术来阐明蜕皮激素生物合成途径几乎没有取得进展，因为中间化合物的量非常低并且极不稳定。几乎所有尝试过这一壮举的实验室在多年的负面结果后都放弃了。首席研究员与明尼苏达大学奥康纳实验室合作，在阐明胆固醇和蜕皮激素之间的生物合成途径方面取得了关键进展。在这些研究中，采用了果蝇果蝇，因为它的遗传学比任何其他动物都更广为人知，而且它的生命周期短使得分子遗传学和生物化学更加可行。分子遗传学和生化分析的结合使用导致了蜕皮激素生物合成途径中两种酶的鉴定、克隆和功能基因组研究，并为阐明该途径中剩余酶的实验方法提供了见解。通过对果蝇胚胎中蜕皮激素含量低于正常水平的某些基因（万圣节基因）进行克隆和测序，首席研究员和他的同事们了解到，这些基因的突变会导致胚胎死亡。将这些基因转染到果蝇细胞系中并使用生化技术表明，其中两个突变体（无实体突变体和影子突变体）分别编码线粒体蜕皮甾醇 22- 和 2- 羟化酶。未来几年继续工作的目的将是通过更复杂的分析（质谱）完全鉴定这两种酶，并鉴定该生物合成途径中剩余的 (5) 种酶（P450 酶）。这些基因的表达将在果蝇发育过程中进行检查，并将在其他昆虫模型系统（例如烟草天蛾）中寻找对应基因。这些实验不仅将全面了解蜕皮激素的生物合成方式，而且还将提供用于转染具有重要农业意义的植物的基因。此外，这些数据应该可以清楚地了解调节蜕皮激素生物合成的限速控制机制，并且也许可以识别由脑神经肽、促胸腺激素控制的特定生物合成反应。这种将分子遗传学与生化分析相结合的新范式将来可用于研究包括病原体在内的多种生物体的生物合成途径。