RESUMO
WSB-1 is a SOCS-box-containing WD-40 protein of unknown function that is induced by Hedgehog signalling in embryonic structures during chicken development. Here we show that WSB-1 is part of an E3 ubiquitin ligase for the thyroid-hormone-activating type 2 iodothyronine deiodinase (D2). The WD-40 propeller of WSB-1 recognizes an 18-amino-acid loop in D2 that confers metabolic instability, whereas the SOCS-box domain mediates its interaction with a ubiquitinating catalytic core complex, modelled as Elongin BC-Cul5-Rbx1 (ECS(WSB-1)). In the developing tibial growth plate, Hedgehog-stimulated D2 ubiquitination via ECS(WSB-1) induces parathyroid hormone-related peptide (PTHrP), thereby regulating chondrocyte differentiation. Thus, ECS(WSB-1) mediates a mechanism by which 'systemic' thyroid hormone can effect local control of the Hedgehog-PTHrP negative feedback loop and thus skeletogenesis.
Assuntos
Lâmina de Crescimento/metabolismo , Proteína Relacionada ao Hormônio Paratireóideo/metabolismo , Proteínas/fisiologia , Hormônios Tireóideos/metabolismo , Transativadores/fisiologia , Ubiquitina-Proteína Ligases/fisiologia , Sequência de Aminoácidos , Animais , Sítios de Ligação/genética , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Embrião de Galinha , Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , Elonguina , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/genética , Lâmina de Crescimento/embriologia , Proteínas Hedgehog , Humanos , Imunoprecipitação , Hibridização In Situ , Peptídeos e Proteínas de Sinalização Intracelular , Iodeto Peroxidase/genética , Iodeto Peroxidase/metabolismo , Camundongos , Modelos Biológicos , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Ligação Proteica , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Subunidades Proteicas/fisiologia , Proteínas/genética , Proteínas/metabolismo , Interferência de RNA , Homologia de Sequência de Aminoácidos , Hormônios Tireóideos/farmacologia , Tíbia/citologia , Tíbia/efeitos dos fármacos , Tíbia/metabolismo , Transativadores/genética , Transativadores/farmacologia , Fatores de Transcrição/metabolismo , Transfecção , Técnicas do Sistema de Duplo-Híbrido , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Contendo Repetições de beta-Transducina/genética , Iodotironina Desiodinase Tipo IIRESUMO
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.
Assuntos
Endopeptidases/fisiologia , Iodeto Peroxidase/fisiologia , Hormônios Tireóideos/biossíntese , Proteínas Supressoras de Tumor , Ubiquitina-Proteína Ligases , Ubiquitina/metabolismo , Adipócitos/metabolismo , Tecido Adiposo Marrom/metabolismo , Western Blotting , Catálise , Catecolaminas/farmacologia , Linhagem Celular , Endopeptidases/metabolismo , Retículo Endoplasmático/metabolismo , Biblioteca Gênica , Humanos , Ligases/metabolismo , Microscopia de Fluorescência , Modelos Biológicos , Plasmídeos/metabolismo , Ligação Proteica , Frações Subcelulares , Temperatura , Tiroxina/metabolismo , Fatores de Tempo , Tri-Iodotironina/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Ubiquitina Tiolesterase , Proteína Supressora de Tumor Von Hippel-Lindau , Iodotironina Desiodinase Tipo IIRESUMO
The goal of the present investigation was to test the hypothesis that types 1, 2, and 3 iodothyronine selenodeiodinases (D1, D2, and D3) can form homodimers. The strategy included transient coexpression of wild-type (wt) deiodinases (target), and FLAG-tagged alanine or cysteine mutants (bait) in human embryonic kidney epithelial cells. SDS-PAGE of the immunoprecipitation pellet of (75)Se-labeled cell lysates using anti-FLAG antibody revealed bands of the correct sizes for the respective wt enzymes, which corresponded to approximately 2-5% of the total deiodinase protein in the cell lysate. Western blot analysis with anti-FLAG antibody of lysates of cells transiently expressing individual FLAG-tagged-cysteine deiodinases revealed specific monomeric bands for each deiodinase and additional minor bands of relative molecular mass (M(r)) of 55,000 for D1, M(r) 62,000 for D2, and M(r) 65,000 for D3, which were eliminated by 100 mM dithiothreitol at 100 C. Anti-FLAG antibody immunodepleted 10% of D1 and 38% of D2 activity from lysates of cells coexpressing inactive FLAG-tagged Ala mutants and the respective wt enzymes (D1 or D2) but failed to immunodeplete wtD3 activity. D1 or D2 activities were present in these respective pellets. We conclude 1) that overexpressed selenodeiodinases can homodimerize probably through disulfide bridges; and 2) at least for D1 and D2, monomeric forms are catalytically active, demonstrating that only one wt monomer partner is required for catalytic activity of these two deiodinases.