Neuropeptide Control of Ecdysone Biosynthesis

为了使昆虫生长和经历变态，它们必须摆脱限制性的外皮（外骨骼）。在数亿年的时间里，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的时候生长。在蜕皮过程中发生的许多结构、生理、生化和分子事件是由昆虫的主要蜕皮激素引起的，多羟基化类固醇激素20-羟基蜕皮激素，其在昆虫的许多组织中由另一种类固醇蜕皮激素合成。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，促前胸腺激素，由昆虫大脑中的四种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中的各个生化步骤方面几乎没有进展。了解合成步骤不仅对我们进一步了解这个密切影响人类福祉的非常重要的动物群体很重要，而且因为如果能够描述蜕皮激素生物合成途径中的各个步骤，并克隆介导这些步骤的酶的基因，有可能将该基因序列导入（转染）农业上重要的植物。然后它们会合成这种类固醇激素，这是一种对害虫的天然威慑。因此，当这种激素被食用这种转染植物的昆虫摄取时，它将脱离同步蜕皮，并且将不能存活。蜕皮激素对高等生物是无毒的，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。由于蜕皮激素合成的中间产物含量低且极不稳定，利用经典的生物化学技术来阐明蜕皮激素合成途径的研究进展甚微。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。在与明尼苏达大学奥康纳实验室的合作中，主要研究者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，黑腹果蝇被采用，因为它的遗传学比任何其他动物都更为人所知，而且它的短生命周期使分子遗传学和生物化学更可行。这种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。通过克隆和测序某些基因（万圣节基因），从果蝇胚胎中含有少于正常量的蜕皮激素，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的致命性。将这些基因转染到果蝇细胞系中并使用生物化学技术已经表明，这些突变体中的两个，离体和阴影，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是通过更复杂的分析（质谱法）充分鉴定这两种酶，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中进行搜索，如烟草天蛾。这些实验不仅将导致蜕皮激素是如何生物合成的完整知识，但他们也将提供基因转染到植物的农业重要性。此外，这些数据应该提供一个清晰的理解的限速控制机制，调节蜕皮激素的生物合成，也许将允许识别特定的生物合成反应，由脑神经肽，prothoracotropic激素控制。这种将分子遗传学与生物化学分析相结合的新范式可在未来用于研究包括病原体在内的各种生物体的生物合成途径。

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.