Deubiquitination of type 2 iodothyronine deiodinase by von Hippel–Lindau protein–interacting deubiquitinating enzymes regulates thyroid hormone activation
Cyntia Curcio-Morelli, … , Guan Wu, Antonio C. Bianco
Cyntia Curcio-Morelli, … , Guan Wu, Antonio C. Bianco
Published July 15, 2003
Citation Information: J Clin Invest. 2003;112(2):189-196. https://doi.org/10.1172/JCI18348.
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Article Endocrinology

Deubiquitination of type 2 iodothyronine deiodinase by von Hippel–Lindau protein–interacting deubiquitinating enzymes regulates thyroid hormone activation

Abstract

The type 2 iodothyronine deiodinase (D2) is an integral membrane ER-resident selenoenzyme that activates the pro-hormone thyroxine (T4) and supplies most of the 3,5,3′-triiodothyronine (T3) that is essential for brain development. D2 is inactivated by selective conjugation to ubiquitin, a process accelerated by T4 catalysis and essential for the maintenance of T3 homeostasis. A yeast two-hybrid screen of a human-brain library with D2 as bait identified von Hippel–Lindau protein–interacting deubiquitinating enzyme-1 (VDU1). D2 interaction with VDU1 and VDU2, a closely related deubiquitinase, was confirmed in mammalian cells. Both VDU proteins colocalize with D2 in the ER, and their coexpression prolongs D2 half-life and activity by D2 deubiquitination. VDU1, but not VDU2, is markedly increased in brown adipocytes by norepinephrine or cold exposure, further amplifying the increase in D2 activity that results from catecholamine-stimulated de novo synthesis. Thus, deubiquitination regulates the supply of active thyroid hormone to brown adipocytes and other D2-expressing cells.

Authors

Cyntia Curcio-Morelli, Ann Marie Zavacki, Marcelo Christofollete, Balazs Gereben, Beatriz C.G. de Freitas, John W. Harney, Zaibo Li, Guan Wu, Antonio C. Bianco

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Figure 2

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VDU1 and VDU2 are ER-resident integral membrane proteins and colocalize ...
VDU1 and VDU2 are ER-resident integral membrane proteins and colocalize with D2. (a) Subcellular fractionation of cell lysates transiently expressing GFP-VDU1, GFP-VDU2, or D10. Fractions of total cell lysate, particulate fraction, and cytosol were isolated and analyzed by Western blot using anti-GFP antibody. This experiment was performed twice. (b) Colocalization of VDU1 (top panels) and VDU2 (bottom panels) with D2. Cells transiently expressing the indicated pairs of proteins were permeabilized with Triton X-100 and processed for immunocytochemistry with anti-FLAG antibody. Visualization was achieved with goat anti-mouse Texas red F(ab′)2 fragment. D2 is shown in red, while the VDUs are shown in green. The yellow bar indicates 10 μm. The distribution spectrum of image pixels is shown in the insets. (c) D2 activity in HEK-293 cells transiently coexpressing D2 and/or VDU1 or VDU2. Cells were harvested 48 hours after transfection. *P < 0.05 vs. D10-transfected cells. (d) D2 activity, as in c except that cells were treated with 0.2% DMSO (vehicle) or 100 μM cycloheximide (CX) and harvested 2 hours later. *P < 0.05 vs. DMSO-treated cells. Values in c and d are the mean ± SD of two to four cell plates; experiments were performed two to three times.