Neuropeptide Control of Ecdysone Biosynthesis

University Of North Carolina At Chapel Hill, Chapel Hill NC

Investigators

Abstract

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.