Global Metabolomics-Based Identification Of Small-Molecule Signals That Regulate Development In Nematodes
Small-molecule signaling serves important functions at all levels of organismal organization and requires diverse biosynthetic mechanisms for encoding biological information in chemical structures. Whereas fungi and bacteria have dedicated biosynthetic machinery that enables production of a great diversity of chemical structures, e.g. polyketides and non-ribosomal peptides, most animals are presumed to lack the ability to produce elaborate small-molecule architectures. Herein, the author describes the integrated use of 2D NMR and high resolution HPLC-MS/MS to aid global metabolomics of complex natural samples. Applications of this methodology to metabolomes of Caenorhabditis elegans and Pristionchus pacificus showed that these nematodes generate a library of complex signaling molecules, ascarosides and paratosides, via selective assembly of building blocks from several primary metabolic pathways, including an unusual xylopyranose -based nucleoside. These compounds act as interorganismal signals controlling larval development, adult morphology, or function as potent attraction pheromones. These findings further indicate species-specific evolution of chemical signaling in nematodes, with regard to both chemical structures and their biological functions. The library of small molecule signals presented in this dissertation, provide striking examples for combinatorial generation of structural diversity in nematodes and connect primary metabolism to regulation of development and adult phenotypic plasticity. Further the author used comparative metabolomics to investigate ascaroside biogenesis in C. elegans. Profiling ascarosides in C. elegans wild-type and peroxisomal -oxidation mutant metabolomes via HPLC-MS/MS and 2D NMR clarified the functions of the acyl-CoA-oxidase, ACOX-1, and the [beta]-ketoacyl-CoA thiolase, DAF-22 in ascaroside biogenesis. Following peroxisomal [beta]-oxidation, the ascarosides are selectively derivatized with moieties of varied biogenetic origin that can dramatically affect biological activity. Finally, using a 2D NMR-based comparative metabolomics approach, the author identified the endogenous ligands of the C. elegans nuclear hormone receptor (NHR), DAF-12, a vitamin D and liver X receptor homolog that regulates larval development, fat metabolism, and lifespan. The identified molecules include only one of two previously predicted DAF-12 ligands and feature unusual structural motifs, e.g. a 1-desaturated steroid. These results demonstrate the advantages of comparative metabolomics over traditional candidate-based approaches and provide a blueprint for the identification of ligands for other C. elegans and mammalian NHRs.
model organism; pheromones; natural products
Lee, Siu Sylvia; Crane, Brian; Brady, Sean Fielding
Chemistry and Chemical Biology
Ph.D. of Chemistry and Chemical Biology
Doctor of Philosophy
dissertation or thesis