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Substrate-Induced Down-Regulation of Human Type 2 Deiodinase (hD2) Is Mediated through Proteasomal Degradation and Requires Interaction with the Enzyme’s Active Center¹

Thyroid Division, Brigham and Women’s Hospital, Harvard Medical School, Boston Massachusetts 02115

Address all correspondence and requests for reprints to: P. Reed Larsen, M.D., Brigham and Women’s Hospital, Thyroid Division—HIM 550, 77 Avenue Louis Pasteur; Boston, Massachusetts 02115. E-mail: Larsen{at}rascal.med.harvard.edu

Type 2 iodothyronine deiodinase (D2) catalyzes the first step in thyroid hormone action, the deiodination of T₄ to T₃ . Endogenous D2 activity is posttranslationally regulated by substrate that accelerates its degradation through the ubiquitin-proteasome pathway. To understand how D2 activity correlates with D2 protein during its normal decay and rT₃-induced down-regulation, HEK-293 cells, transiently expressing human D2, were labeled with Na⁷⁵SeO₃ and then treated with 100 µM cycloheximide (CX), 30 nM rT₃, and/or 10 µM MG132, a specific proteasome inhibitor, for 2–4 h. D2 protein and enzyme activity changed in parallel, disappearing with a half-life of 2 h in the presence of CX, or 1 h when CX + rT₃ were combined. Treatment with MG132 blocked these effects. We created selenocysteine (Sec) 133 to cysteine (Cys) or alanine (Ala) D2 mutants, without changing Sec 266. The CysD2 activity and protein levels were also parallel, with a similar half-life of approximately 2 h, whereas the rT₃-induced D2 down-regulation required approximately 1000-fold higher rT₃ concentration (30 µM) due to a proportionally higher Michaelis constant of CysD2. In similar experiments, the AlaD2 mutant retained the short half-life but was not catalytically active and not susceptible to rT₃-accelerated degradation. We conclude that substrate-induced loss of D2 activity is due to proteasomal degradation of the enzyme and requires interaction with the catalytic center of the protein.

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