Thyroid hormones exert profound and diverse effects on multiple tissues in both developing and adult animals, including humans. Thyroxine (T4), the principal secretory product of the thyroid gland, functions primarily as a prohormone. In peripheral tissues, T4 is deiodinated at the 5'-position forming the metabolically active hormone 3,5,3'-triiodothyronine (T3), which in turn is metabolized to inactive compounds by the process of 5- deiodination. These metabolic processes are catalyzed by three deiodinases, termed types I, II, and III, whose properties and patterns of expression are distinctive, allowing for tissue specificity in the control of thyroid hormone action. Such regulatory control of thyroid hormone metabolism appears to be of particular importance in the fetoplacental unit and during states of illness, altered nutritional status, and thyroid dysfunction. Despite progress in the understanding of certain aspects of thyroid hormone metabolism, including our recent characterization of a cDNA that encodes a selenoprotein with type III deiodinase (5DIII) activity, several critical aspects of these processes remain poorly defined, including the biochemical structure and functional correlates of the deiodinases, their exact physiologic role in regulating thyroid hormone action, and the intracellular processes controlling their activity. The present proposal addresses these questions within the context of a long range goal of defining the physiologic and biochemical parameters mediating thyroid hormone action and metabolism.
The specific aims of this proposal are: (1) To define structural domains of the 5'DI and 5DIII that dictate substrate specificity and susceptibility to inhibition by propylthiouracil and aurothioglucose. This will be accomplished by site-directed mutagenesis and the construction of chimeric deiodinase proteins. (2) To isolate and characterize cDNAs encoding the rat 5DIII and 5'DII by PCR-based and filter hybridization cloning strategies. (3) To determine the functional significance and influence of selenium status on the relative proportions of two 5'DI mRNAs present in rat liver and kidney. These studies will extend our recent observation that intracellular processing of 5'DI mRNA in rat liver and kidney appears to involve the use of alternative polyadenylation sites, with the relative abundance of the resultant transcripts influenced significantly by the selenium status of the animal. (4) To investigate, by cross-linking studies and site-directed mutagenesis, the role of thioredoxin, protein disulfide isomerase, and the importance of thioldisulfide exchange in the catalytic mechanism and regulation of the rat 5'DI. (5) To investigate the physiologic significance of the high levels of 5DIII activity present in rat skin and the effects of thyroid hormone on gene expression in this tissue. These studies will lay the groundwork for a more complete understanding of the important role of deiodination and selenium in the regulation of cellular metabolic processes and thyroid hormone action.
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