Substrate-induced down-regulation of human type 2 deiodinase (hD2) is mediated through proteasomal degradation and requires interaction with the enzyme's active center

TY - JOUR

T1 - Substrate-induced down-regulation of human type 2 deiodinase (hD2) is mediated through proteasomal degradation and requires interaction with the enzyme's active center

AU - Steinsapir, Jaime

AU - Bianco, Antonio C.

AU - Buettner, Christoph

AU - Harney, John

AU - Larsen, P. R.

PY - 2000

Y1 - 2000

N2 - 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.

AB - 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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U2 - https://doi.org/10.1210/endo.141.3.7355

DO - https://doi.org/10.1210/endo.141.3.7355

M3 - Article

C2 - 10698189

SN - 0013-7227

VL - 141

SP - 1127

EP - 1135

JO - Endocrinology

JF - Endocrinology

IS - 3

ER -