Thyroid hormone action begins with the deiodination of thyroxine (T4) to 3,5,3'-triiodothyronine (T3). Two enzymes have been identified which catalyze this reaction; Types I and II iodothyronine deiodinases (5'DI and 5'DII). The long-term objectives of this application are to understand the regulation of T4 activation in animals and man. We have recently cloned a 2.1 kb cDNA for the rat 5'DI enzyme and found it contains the rare amino acid, selenocysteine, which is critical to its normal catalytic function. There are two Specific Aims in the present proposal. The first is to identify factors which influence the catalytic potency and tissue content of the 5'DI. Four aspects of this enzyme and its synthesis will be explored. 1) Structure/function relationships will be analyzed by in vitro expression of mutants with selected amino acid alterations. Catalytic activity and BrAc[125I]T4 labeling will be compared. 2) Translational regulation, especially of those steps required for UGA-directed cotranslational selenocysteine insertion., will be studied in 5'DI expressing cell lines and transiently transfected COS-7 and JEG-3 cells. 3) The mechanism of hormonal and nutritional regulation of 5'DI mRNA levels will be evaluated in mice and rats and in 5'DI expressing cell lines (GC,LLC-PK1,FRTL-5). 4) Trans and cis acting factors influencing 5'DI promoter function will be identified with special emphasis on comparing 5'DI in benign and malignant human thyroid tumors and in an animal model of inherited deficiency of 5'DI expression. The second Specific Aim will be to clone the 5'DII and compare its structure, function, and regulation with that of the 5'DI enzyme. We have developed chromatographic strategies which result in 2700 fold purification of active 5'DII from BAT with greater than 20% yield. These will be combined with BrAc[l25I]T4 labeling to isolate the 29 kD catalytic subunit for either microsequencing or antibody production. BAT cDNA libraries will be screened either with sequence-based degenerate oligos, specific antibody or BrAc[125I]T4. These studies will provide novel insights into the basic processes regulating thyroid hormone activation, as well as new information about the factors controlling the synthesis of selenocysteine containing proteins in eukaryotes. The presence or absence of 5'DI in thyroid nodule aspirates could prove to be a useful criteria for differentiating benign from malignant tumors.
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