Thyroid hormones are of unquestionable importance in regulating metabolic processes in all tissues. Thyroxine (T4), the principal secretory product of the thyroid gland, functions primarily as a prohormone and is converted in extrathyroidal tissues to the metabolically more active 3,3',5- triiodothyronine (T3) and the largely inactive 3,3',5-triiodothyronine (rT3) by removal of an iodine from the 5'- or 5-position, respectively. Because the majority of thyroid hormone effects are mediated through the interaction of T3 with nuclear receptors, the regulatory control of T4 and T3 deiodination is critically important to thyroid hormone action. The long range goal of this project is to determine the biochemical mechanisms and physiologic parameters which regulate the metabolism of thyroid hormones. Studies are focused specifically on defining the cellular mechanisms regulating the activity of the iodothyronine deiodinases, the family of enzymes which convert T4 to T3 and rT3 in extrathyroidal tissues. The regulation of these enzymes is hormone- dependent and tissue-specific, and involves complex alterations in enzyme activation and inactivation. To date the deiodinase enzymes have proved difficult to purify, and thus little is known about their biochemical characteristics or the mechanisms which underlie alterations in their activity. Presented in this proposal are a detailed set of experiments designed to delineate the pre- and post-translational biochemical mechanisms mediating changes in deiodinase activity.
The specific aims of this project include: (1) to isolate and characterize a cDNA encoding the type I 5'-deiodinase (5'DI) by utilizing Xenopus laevis oocytes as a in vivo translational assay system for screening a cDNA library; (2) to determine the biochemical characteristics of the translated 5'DI encoded by the isolated cDNA; (3) to detail the tissue distribution and regulation of 5'DI mRNA using molecular hybridization techniques; (4) to determine the effects of ligands, propylthiouracil, and sulfhydryl reagents on the inactivation of types I, II, and III iodothyronine deiodinases; (5) to investigate the mechanisms whereby thyroid hormones, ligands and sulfhydryl reagents regulate 5'DI by producing specific antibodies against this enzyme and using these antibodies in immunoblotting and immunoprecipitation experiments; (6) to isolate and characterized cDNAs which encode the type II 5'-deiodinase and the type III 5-deiodinase enzymes. The studies proposed here will provide significant insight into the biochemical characteristics of the deiodinase enzymes and the complex pre- and post-translational regulatory processes controlling their activity. Furthermore, the availability of cDNAs and specific antibodies for these enzymes will dramatically enhance our ability to investigate many fundamental issues in thyroid hormone biochemistry and physiology. Thus, these studies will provide a framework for analyzing and further investing the alterations in thyroid hormone economy which occur frequently in clinical medicine.
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