Deubiquitination of type 2 iodothyronine deiodinase by von Hippel–Lindau protein–interacting deubiquitinating enzymes regulates thyroid hormone activation
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
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
View: Text | PDF
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

×

Figure 1

Options: View larger image (or click on image) Download as PowerPoint
Yeast two-hybrid mating for pGBKT7-D2 and VDU1-expressing clone. (a and ...
Yeast two-hybrid mating for pGBKT7-D2 and VDU1-expressing clone. (a and b) Positive control interaction for the mating: AH109 (pGBKT7-53) plus Y187 (pTD1); negative control for the mating: AH109 (pGBKT7-empty) plus Y187 (pGADT7-empty); mating: AH109 (pGBKT7-D2) plus Y187 (pGADT7-VDU1); negative control for the VDU1 mating: AH109 (pGBKT7-empty) plus Y187 (pGADT7-VDU1). (a) Mating grown on Trp–Leu– double-dropout (DDO) media. (b) Mating grown on Trp–Leu–His–Ade– QDO media plus X-α-Gal substrate. While all yeast strains grew on DDO media, indicating the presence of both expression plasmids, D2 did not interact with the GAL4 AD alone (data not shown), and, on QDO media (b), VDU1 did not interact with the GAL4 DBD alone, while pGBKT7-D2 interacted with the pGADT7-VDU1 clone. The top left panel indicates the growth of the positive control, while the negative control is indicated in the top right panel. The growth of PGBKT7-D2 + VDU-1 on QDO media plus X-α-Gal is shown on the lower left panel, while pGBKT7-empty + VDU-1 is shown on the lower right panel, indicating specificity of VDU-1 interaction with D2. (c) In vitro coimmunoprecipitation of GST-D2 and 35S-VDU1. Crude bacterial lysates expressing GST-D2 or GST-GUS were incubated with 35S-VDU1 followed by GST pulldown. Pellets were resolved by SDS-PAGE, and 35S-VDU1 is indicated. Levels of GST-fusion proteins (GST-D2 and GST-GUS) were determined by Western analysis using anti-GST antibody. Approximately 1% of the input 35S-VDU1 is specifically pulled down. This experiment was performed twice. (d) In vivo coimmunoprecipitation of D2 and VDU1 or VDU2. HEK-293 cells were cotransfected with FLAG-CysD2 and GFP-VDU1 or GFP-VDU2. Anti-GFP antibody was used for immunoprecipitation, and the pellets were resolved by SDS-PAGE and probed with anti-FLAG antibody by Western analysis. GFP refers to a vector containing only GFP, not fused to any other protein. This experiment was performed twice with similar results.