SELENODEIODINASE PROCESSING BY THE PROTEASOME SYSTEM

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DESCRIPTION: (Adapted from the applicant's abstract) This is a new proposal to determine the mechanisms regulating the rate-limiting steps in the ubiquitin proteasome system leading to the controlled degradation of the Types 1, 2 and 3 selenodeiodinases. D2 is a critical enzyme regulating the first step in thyroid hormone action, the conversion of the prohormone T4 to the active hormone, T3. This process occurs intracellularly in brain, pituitary and brown fat in which the T3 produced constitutes a major fraction of the nuclear receptor-bound hormone. Furthermore, it appears that, in humans, unlike in adult rodents, D2 may also generate a significant fraction of plasma T3 by virtue of its wide tissue expression in skeletal muscle. It has been known for a number of years that post-translational regulation of D2 by substrate is a major regulatory step in determining the tissue level of the active enzyme. Recent studies have shown that degradation of D2 occurs in proteasomes, that its half-life is quite short (<1 hr), that its degradation is accelerated 2-fold by exposure to substrate and that this process requires interaction of substrate with a D2 enzyme which has either selenocysteine or cysteine in its active center. To define the regulatory steps in this process, the investigator will use several complementary systems. These include transient expression in HEK-293 cells and a temperature-sensitive CHO cell line containing a mutant UB-1 enzyme. A third strategy will be to exploit the fact that D2 can be expressed in yeast with well-documented mutations in the ubiquitin-proteasome degradation pathway and that it is possible to express both wild type and epitope-labeled protein to evaluate changes in activity those in D2 protein levels. Lastly, the investigator will employ cell-free systems which will allow the study of specific components of the ubiquitin proteasome system in the metabolism of D2 and compare results with those for Dl and D3 which have significantly longer half-lives. From a physiological perspective, it is quite conceivable that the accelerated proteolysis of D2 in human skeletal muscle during starvation or illness could explain the rapid onset of the "low T3 syndrome." This is a well-recognized but poorly understood phenomenon which occurs in every human under food restriction or with significant systemic illness.