| Small molecules, such as steroid hormones, are key regulators of intracellular communication. Steroid hormones exert their action by binding to nuclear receptors in target organs, allowing nuclear receptors to translate endocrine signals into physiological outputs by regulating the transcription of target genes. My thesis has focused on two poorly understood aspects of physiological regulation by small molecules. In Chapter 1, I investigated the mechanism by which cholesterol-derived steroid hormones control nuclear receptor function in mammalian cells, focusing on the regulation of the glucocorticoid receptor (GR) intracellular localization by the steroid hormone corticosterone. Binding corticosterone changes the localization of GR from the cytoplasm to the nucleus. Using the genetics of S. cerevisiae, I identified an import receptor, Sxm1, critical for GR nuclear import in yeast. Two metazoan homologues of Sxm1, importin 7 and importin 8, can bind GR, and importin 7 can import a fragment of GR in an in vitro nuclear import assay. Interestingly, importin 7 and importin 8 bound GR even in the absence of hormone, suggesting that hormonal control of localization lies downstream of import receptor binding.; In Chapter 2, I used C. elegans to probe the roles of small molecules in regulating physiogical processes. In the absence of cholesterol, worms display defects in growth, reproduction, molting, dauer formation, and gonandal migration (mig). As cholesterol serves as a precursor for a subset of nuclear receptor ligands in mammalian cells, I tested in worms the effects of inhibitors of mammalian cholesterol metabolism. Worms grown in the presence of ketoconazole displayed growth, mig, and reproductive defects. I determined that the mig but not the growth and reproductive phenotypes required the function of a nuclear receptor, daf-12, suggesting that ketoconazole may block the formation of a cholesterol-derived ligand for daf-12 . These data suggest that ketoconazole affects multiple C. elegans signaling pathways and will serve as a valuable tool for investigating the molecular role of cholesterol in C. elegans physiology. Lessons learned from these experiments will also likely be relevant to more complex metazoans, such as mammals. |
