Neuropeptide Control of Ecdysone Biosynthesis

为了使昆虫生长和变形，它们必须蜕去限制性的外皮（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害环境因素威胁较小的时候生长。昆虫在蜕皮过程中发生的许多结构、生理、生化和分子事件都是由一种主要的蜕皮激素引起的，这种激素是一种多羟基类固醇激素20-羟基蜕皮激素，它是由另一种类固醇激素蜕皮激素在昆虫的许多组织中合成的。蜕皮素是在昆虫的特殊腺体——前胸腺中合成的。刺激合成这种重要分子的是一种肽，一种促胸激素，由昆虫大脑中四个专门的神经分泌细胞分泌。大约40年前，蜕皮激素被从两吨商品蚕中提取并鉴定，但在过去的40年里，蜕皮激素合成的单个生化步骤的阐明几乎没有进展。了解这些合成步骤不仅对我们进一步了解这种密切影响人类福祉的重要动物群体很重要，而且因为如果我们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，就有可能将这些基因序列（转染）引入重要的农业植物中。然后他们会合成这种类固醇激素，这是一种天然的害虫威慑。因此，当这种激素被食用这种转染植物的昆虫摄入时，它就会脱皮不同步，无法存活。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。由于蜕皮激素的中间产物数量少且极不稳定，传统的生物化学技术对蜕皮激素生物合成途径的研究进展甚微。几乎所有尝试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，首席研究员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，果蝇黑腹果蝇（Drosophila melanogaster）之所以被使用，是因为它的遗传学比其他任何动物都更为人所知，而且它的生命周期短，使得分子遗传学和生物化学都更可行。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。通过对果蝇胚胎中蜕皮激素含量低于正常水平的特定基因（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的致命性。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将是通过更复杂的分析（质谱分析）充分鉴定这两种酶，并鉴定这种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测，并将在其他昆虫模型系统（如烟草角虫）中寻找相应的基因。这些实验不仅使我们全面了解蜕皮激素是如何生物合成的，而且还将为转染到重要的农业植物中提供基因。此外，这些数据应该提供对调节蜕皮激素生物合成的限速控制机制的清晰理解，并且可能允许识别由脑神经肽，促胸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模式可以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.