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
While bile acids (BAs) have long been known to be essential in dietary lipid absorption and cholesterol catabolism, in recent years an important role for BAs as signalling molecules has emerged. BAs activate mitogen-activated protein kinase pathways, are ligands for the G-protein-coupled receptor (GPCR) TGR5 and activate nuclear hormone receptors such as farnesoid X receptor alpha (FXR-alpha; NR1H4). FXR-alpha regulates the enterohepatic recycling and biosynthesis of BAs by controlling the expression of genes such as the short heterodimer partner (SHP; NR0B2) that inhibits the activity of other nuclear receptors. The FXR-alpha-mediated SHP induction also underlies the downregulation of the hepatic fatty acid and triglyceride biosynthesis and very-low-density lipoprotein production mediated by sterol-regulatory-element-binding protein 1c. This indicates that BAs might be able to function beyond the control of BA homeostasis as general metabolic integrators. Here we show that the administration of BAs to mice increases energy expenditure in brown adipose tissue, preventing obesity and resistance to insulin. This novel metabolic effect of BAs is critically dependent on induction of the cyclic-AMP-dependent thyroid hormone activating enzyme type 2 iodothyronine deiodinase (D2) because it is lost in D2-/- mice. Treatment of brown adipocytes and human skeletal myocytes with BA increases D2 activity and oxygen consumption. These effects are independent of FXR-alpha, and instead are mediated by increased cAMP production that stems from the binding of BAs with the G-protein-coupled receptor TGR5. In both rodents and humans, the most thermogenically important tissues are specifically targeted by this mechanism because they coexpress D2 and TGR5. The BA-TGR5-cAMP-D2 signalling pathway is therefore a crucial mechanism for fine-tuning energy homeostasis that can be targeted to improve metabolic control.
Assuntos
Ácidos e Sais Biliares/farmacologia , Metabolismo Energético/efeitos dos fármacos , Hormônios Tireóideos/metabolismo , Tecido Adiposo Marrom/efeitos dos fármacos , Tecido Adiposo Marrom/enzimologia , Tecido Adiposo Marrom/metabolismo , Adiposidade/efeitos dos fármacos , Animais , Peso Corporal/efeitos dos fármacos , Dióxido de Carbono/metabolismo , Ácido Cólico/farmacologia , AMP Cíclico/biossíntese , Gorduras na Dieta/administração & dosagem , Gorduras na Dieta/farmacologia , Deleção de Genes , Homeostase/efeitos dos fármacos , Humanos , Iodeto Peroxidase/deficiência , Iodeto Peroxidase/genética , Iodeto Peroxidase/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Células Musculares/efeitos dos fármacos , Células Musculares/enzimologia , Células Musculares/metabolismo , Músculo Esquelético/citologia , Consumo de Oxigênio/efeitos dos fármacos , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Iodotironina Desiodinase Tipo IIRESUMO
The FoxO3-dependent increase in type II deiodinase (D2), which converts the prohormone thyroxine (T(4)) to 3,5,3'-triiodothyronine (T(3)), is required for normal mouse skeletal muscle differentiation and regeneration. This implies a requirement for an increase in D2-generated intracellular T(3) under these conditions, which has not been directly demonstrated despite the presence of D2 activity in skeletal muscle. We directly show that D2-mediated T(4)-to-T(3) conversion increases during differentiation in C(2)C(12) myoblast and primary cultures of mouse neonatal skeletal muscle precursor cells, and that blockade of D2 eliminates this. In adult mice given (125)I-T(4) and (131)I-T(3), the intracellular (125)I-T(3)/(131)I-T(3) ratio is significantly higher than in serum in both the D2-expressing cerebral cortex and the skeletal muscle of wild-type, but not D2KO, mice. In D1-expressing liver and kidney, the (125)I-T(3)/(131)I-T(3) ratio does not differ from that in serum. Hypothyroidism increases D2 activity, and in agreement with this, the difference in (125)I-T(3)/(131)I-T(3) ratio is increased further in hypothyroid wild-type mice but not altered in the D2KO. Notably, in wild-type but not in D2KO mice, the muscle production of (125)I-T(3) is doubled after skeletal muscle injury. Thus, D2-mediated T(4)-to-T(3) conversion generates significant intracellular T(3) in normal mouse skeletal muscle, with the increased T(3) required for muscle regeneration being provided by increased D2 synthesis, not by T(3) from the circulation.
Assuntos
Iodeto Peroxidase/fisiologia , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiologia , Regeneração , Tri-Iodotironina/metabolismo , Animais , Animais Recém-Nascidos , Células Cultivadas , Córtex Cerebral/química , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Iodeto Peroxidase/genética , Iodeto Peroxidase/metabolismo , Radioisótopos do Iodo/farmacocinética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mioblastos/química , Mioblastos/efeitos dos fármacos , Mioblastos/metabolismo , Regeneração/fisiologia , Tri-Iodotironina Reversa/farmacologia , Iodotironina Desiodinase Tipo IIRESUMO
Ubiquitination is a critical posttranslational regulator of protein stability and/or subcellular localization. Here we show that ubiquitination can also regulate proteins by transiently inactivating enzymatic function through conformational change in a dimeric enzyme, which can be reversed upon deubiquitination. Our model system is the thyroid hormone-activating type 2 deiodinase (D2), an endoplasmic reticulum-resident type 1 integral membrane enzyme. D2 exists as a homodimer maintained by interacting surfaces at its transmembrane and globular cytosolic domains. The D2 dimer associates with the Hedgehog-inducible ubiquitin ligase WSB-1, the ubiquitin conjugase UBC-7, and VDU-1, a D2-specific deubiquitinase. Upon binding of T4, its natural substrate, D2 is ubiquitinated, which inactivates the enzyme by interfering with D2's globular interacting surfaces that are critical for dimerization and catalytic activity. This state of transient inactivity and change in dimer conformation persists until deubiquitination. The continuous association of D2 with this regulatory protein complex supports rapid cycles of deiodination, conjugation to ubiquitin, and enzyme reactivation by deubiquitination, allowing tight control of thyroid hormone action.
Assuntos
Iodeto Peroxidase/química , Iodeto Peroxidase/metabolismo , Ubiquitina/metabolismo , Sequência de Aminoácidos , Animais , Catálise , Domínio Catalítico , Linhagem Celular , Dimerização , Holoenzimas/química , Holoenzimas/metabolismo , Humanos , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Relação Estrutura-AtividadeRESUMO
Selenocysteine is incorporated into proteins via "recoding" of UGA from a stop codon to a sense codon, a process that requires specific secondary structures in the 3' untranslated region, termed selenocysteine incorporation sequence (SECIS) elements, and the protein factors that they recruit. Whereas most selenoprotein mRNAs contain a single UGA codon and a single SECIS element, selenoprotein P genes encode multiple UGAs and two SECIS elements. We have identified evolutionary adaptations in selenoprotein P genes that contribute to the efficiency of incorporating multiple selenocysteine residues in this protein. The first is a conserved, inefficiently decoded UGA codon in the N-terminal region, which appears to serve both as a checkpoint for the presence of factors required for selenocysteine incorporation and as a "bottleneck," slowing down the progress of elongating ribosomes. The second adaptation involves the presence of introns downstream of this inefficiently decoded UGA which confer the potential for nonsense-mediated decay when factors required for selenocysteine incorporation are limiting. Third, the two SECIS elements in selenoprotein P mRNA function with differing efficiencies, affecting both the rate and the efficiency of decoding different UGAs. The implications for how these factors contribute to the decoding of multiple selenocysteine residues are discussed.
Assuntos
Códon/genética , Biossíntese de Proteínas , Ribossomos/genética , Selenocisteína/metabolismo , Selenoproteína P/genética , Proteínas de Peixe-Zebra/genética , Animais , Linhagem Celular , Códon de Terminação/genética , Evolução Molecular , Humanos , Mutação , Biossíntese de Proteínas/genética , Precursores de RNA/biossíntese , Precursores de RNA/genética , Precursores de RNA/metabolismo , Selenocisteína/genética , Selenoproteína P/biossíntese , Selenoproteína P/metabolismo , Deleção de Sequência , Peixe-Zebra , Proteínas de Peixe-Zebra/biossíntese , Proteínas de Peixe-Zebra/metabolismoRESUMO
Recoding of UGA from a stop codon to selenocysteine poses a dilemma for the protein translation machinery. In eukaryotes, two factors that are crucial to this recoding process are the mRNA binding protein of the Sec insertion sequence, SBP2, and the specialized elongation factor, EFsec. We sought to determine the subcellular localization of these selenoprotein synthesis factors in mammalian cells and thus gain insight into how selenoprotein mRNAs might circumvent nonsense-mediated decay. Intriguingly, both EFsec and SBP2 localization differed depending on the cell line but significant colocalization of the two proteins was observed in cells where SBP2 levels were detectable. We identify functional nuclear localization and export signals in both proteins, demonstrate that SBP2 undergoes nucleocytoplasmic shuttling, and provide evidence that SBP2 levels and localization may influence EFsec localization. Our results suggest a mechanism for the nuclear assembly of the selenocysteine incorporation machinery that could allow selenoprotein mRNAs to circumvent nonsense-mediated decay, thus providing new insights into the mechanism of selenoprotein translation.
Assuntos
Núcleo Celular/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Selenoproteínas/genética , Selenoproteínas/metabolismo , Transporte Ativo do Núcleo Celular , Sequência de Aminoácidos , Animais , Linhagem Celular , Núcleo Celular/genética , Citoplasma/metabolismo , Código Genético , Humanos , Camundongos , Dados de Sequência Molecular , Sinais de Exportação Nuclear , Sinais de Localização Nuclear , Fatores de Alongamento de Peptídeos/genética , Fatores de Alongamento de Peptídeos/metabolismo , Estrutura Terciária de Proteína , Proteínas de Ligação a RNA/genética , Ratos , Selenocisteína/genética , Selenocisteína/metabolismoRESUMO
Selenocysteine incorporation in eukaryotes occurs cotranslationally at UGA codons via the interactions of RNA-protein complexes, one comprised of selenocysteyl (Sec)-tRNA([Ser]Sec) and its specific elongation factor, EFsec, and another consisting of the SECIS element and SECIS binding protein, SBP2. Other factors implicated in this pathway include two selenophosphate synthetases, SPS1 and SPS2, ribosomal protein L30, and two factors identified as binding tRNA([Ser]Sec), termed soluble liver antigen/liver protein (SLA/LP) and SECp43. We report that SLA/LP and SPS1 interact in vitro and in vivo and that SECp43 cotransfection increases this interaction and redistributes all three proteins to a predominantly nuclear localization. We further show that SECp43 interacts with the selenocysteyl-tRNA([Ser]Sec)-EFsec complex in vitro, and SECp43 coexpression promotes interaction between EFsec and SBP2 in vivo. Additionally, SECp43 increases selenocysteine incorporation and selenoprotein mRNA levels, the latter presumably due to circumvention of nonsense-mediated decay. Thus, SECp43 emerges as a key player in orchestrating the interactions and localization of the other factors involved in selenoprotein biosynthesis. Finally, our studies delineating the multiple, coordinated protein-nucleic acid interactions between SECp43 and the previously described selenoprotein cotranslational factors resulted in a model of selenocysteine biosynthesis and incorporation dependent upon both cytoplasmic and nuclear supramolecular complexes.
Assuntos
Complexos Multiproteicos/metabolismo , Proteínas de Ligação a RNA/metabolismo , Selenocisteína/metabolismo , Autoantígenos/genética , Autoantígenos/metabolismo , Núcleo Celular/metabolismo , Células Cultivadas , Códon de Terminação , Citoplasma/metabolismo , Humanos , Fatores de Alongamento de Peptídeos/genética , Fatores de Alongamento de Peptídeos/metabolismo , Fosfotransferases/genética , Fosfotransferases/metabolismo , RNA Mensageiro/metabolismo , RNA de Transferência de Serina/genética , RNA de Transferência de Serina/metabolismo , Proteínas de Ligação a RNA/genética , Selenoproteínas/biossíntese , Selenoproteínas/metabolismoRESUMO
The type 3 deiodinase (D3) inactivates thyroid hormone action by catalyzing tissue-specific inner ring deiodination, predominantly during embryonic development. D3 has gained much attention as a player in the euthyroid sick syndrome, given its robust reactivation during injury and/or illness. Whereas much of the structure biology of the deiodinases is derived from studies with D2, a dimeric endoplasmic reticulum obligatory activating deiodinase, little is known about the holostructure of the plasma membrane resident D3, the deiodinase capable of thyroid hormone inactivation. Here we used fluorescence resonance energy transfer in live cells to demonstrate that D3 exists as homodimer. While D3 homodimerized in its native state, minor heterodimerization was also observed between D3:D1 and D3:D2 in intact cells, the significance of which remains elusive. Incubation with 0.5-1.2 m urea resulted in loss of D3 homodimerization as assessed by bioluminescence resonance energy transfer and a proportional loss of enzyme activity, to a maximum of approximately 50%. Protein modeling using a D2-based scaffold identified potential dimerization surfaces in the transmembrane and globular domains. Truncation of the transmembrane domain (DeltaD3) abrogated dimerization and deiodinase activity except when coexpressed with full-length catalytically inactive deiodinase, thus assembled as DeltaD3:D3 dimer; thus the D3 globular domain also exhibits dimerization surfaces. In conclusion, the inactivating deiodinase D3 exists as homo- or heterodimer in living intact cells, a feature that is critical for their catalytic activities.
Assuntos
Iodeto Peroxidase/metabolismo , Iodeto Peroxidase/fisiologia , Hormônios Tireóideos/metabolismo , Sequência de Aminoácidos , Catálise , Células Cultivadas , Dimerização , Transferência Ressonante de Energia de Fluorescência , Humanos , Iodeto Peroxidase/química , Iodeto Peroxidase/genética , Proteínas Luminescentes/análise , Proteínas de Membrana/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Estrutura Terciária de Proteína/fisiologia , Homologia de Sequência de Aminoácidos , Propriedades de Superfície , TransfecçãoRESUMO
Human type II deiodinase is a master regulator of thyroid hormone activation in several tissues. In placenta, type II deiodinase mRNA levels and enzymatic activity are elevated only during the first trimester of pregnancy and then progressively decline. During this early stage, mitogens such as epidermal growth factor (EGF) have been shown to promote the proliferation of the trophoblast by acting through multiple mechanisms. Here we show that EGF modulates transcription of human type II deiodinase gene (Dio2) through distinct signaling pathways, leading to the assembly of a heterogeneous transcription factor complex. Gene expression and deiodination assays have shown that EGF promptly induces a short-lived Dio2 mRNA and enzymatic activity. The induction is mediated by ERK and p38 kinases, as demonstrated by selective inhibition or overexpression of different mitogen-activated kinases. Reporter assays of mutant constructs indicate that EGF-induced transcriptional activity on Dio2 promoter is mediated by the cAMP response element (CRE) and does not involve the activating protein 1 site. With functional and biochemical approaches, we have demonstrated that the EGF stimulation culminates with the assembly and recruitment over the Dio2 CRE of a composite complex, which consists of c-Jun, c-Fos, and CRE-binding protein. These results further support the hypothesis that placental iodothyronine metabolism is critical during early pregnancy.
Assuntos
Fator de Crescimento Epidérmico/metabolismo , Iodeto Peroxidase/genética , Placenta/citologia , Hormônios Tireóideos/metabolismo , Linhagem Celular Tumoral , Coriocarcinoma , Colforsina/farmacologia , AMP Cíclico/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Fator de Crescimento Epidérmico/farmacologia , Feminino , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/fisiologia , Humanos , Iodeto Peroxidase/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Gravidez , Proteínas Proto-Oncogênicas c-fos/metabolismo , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/fisiologia , Neoplasias Uterinas , Iodotironina Desiodinase Tipo IIRESUMO
Disturbances in energy homeostasis can result in obesity and other metabolic diseases. Here we report a metabolic pathway present in normal human skeletal muscle myoblasts that is activated by the small polyphenolic molecule kaempferol (KPF). Treatment with KPF leads to an approximately 30% increase in skeletal myocyte oxygen consumption. The mechanism involves a several-fold increase in cyclic AMP (cAMP) generation and protein kinase A activation, and the effect of KPF can be mimicked via treatment with dibutyryl cAMP. Microarray and real-time PCR studies identified a set of metabolically relevant genes influenced by KPF including peroxisome proliferator-activated receptor gamma coactivator-1alpha, carnitine palmitoyl transferase-1, mitochondrial transcription factor 1, citrate synthase, and uncoupling protein-3, although KPF itself is not a direct mitochondrial uncoupler. The cAMP-responsive gene for type 2 iodothyronine deiodinase (D2), an intracellular enzyme that activates thyroid hormone (T3) for the nucleus, is approximately threefold upregulated by KPF; furthermore, the activity half-life for D2 is dramatically and selectively increased as well. The net effect is an approximately 10-fold stimulation of D2 activity as measured in cell sonicates, with a concurrent increase of approximately 2.6-fold in the rate of T3 production, which persists even 24 h after KPF has been removed from the system. The effects of KPF on D2 are independent of sirtuin activation and only weakly reproduced by other small polyphenolic molecules such as quercetin and fisetin. These data document a novel mechanism by which a xenobiotic-activated pathway can regulate metabolically important genes as well as thyroid hormone activation and thus may influence metabolic control in humans.
Assuntos
Metabolismo Energético/efeitos dos fármacos , Quempferóis/farmacologia , Tri-Iodotironina/metabolismo , Animais , Linhagem Celular , Chalconas/farmacologia , AMP Cíclico/metabolismo , Proteína Quinase Tipo II Dependente de AMP Cíclico , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Iodeto Peroxidase/genética , Iodeto Peroxidase/metabolismo , Mioblastos/efeitos dos fármacos , Consumo de Oxigênio/efeitos dos fármacos , Interferência de RNA , Ratos , Resveratrol , Estilbenos/farmacologia , Iodotironina Desiodinase Tipo IIRESUMO
The relative roles of the types 1 and 2 iodothyronine deiodinases (D1 and D2) in extrathyroidal 3,5,3'-triiodothyronine (T3) production in humans are unknown. We calculated the rate of thyroxine (T4) to T3 conversion by intact cells transiently expressing D1 or D2 at low (2 pM), normal (20 pM), and high (200 pM) free T4 concentrations. Deiodinase activities were then assayed in cell sonicates. The ratio of T3 production in cell sonicates (catalytic efficiency) was multiplied by the tissue activities reported in human liver (D1) and skeletal muscle (D2). From these calculations, we predict that in euthyroid humans, D2-generated T3 is 29 nmol/d, while that of D1-generated T3 is 15 nmol/d, from these major deiodinase-expressing tissues. The total estimated extrathyroidal T3 production, 44 nmol/d, is in close agreement with the 40 nmol T3/d based on previous kinetic studies. D2-generated T3 production accounts for approximately 71% of the peripheral T3 production in hypothyroidism, but D1 for approximately 67% in thyrotoxic patients. We also show that the intracellular D2-generated T3 has a greater effect on T3-dependent gene transcription than that from D1, which indicates that generation of nuclear T3 is an intrinsic property of the D2 protein. We suggest that impairment of D2-generated T3 is the major cause of the reduced T3 production in the euthyroid sick syndrome.
Assuntos
Iodeto Peroxidase/metabolismo , Glândula Tireoide/metabolismo , Tri-Iodotironina/sangue , Linhagem Celular , Humanos , Iodeto Peroxidase/genética , Radioisótopos do Iodo/metabolismo , Fígado/metabolismo , Estrutura Molecular , Músculo Esquelético/metabolismo , Tiroxina/química , Tiroxina/metabolismo , Tri-Iodotironina/química , 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
T(4), the main product of thyroid secretion, is a critical signal in plasma that mediates the TSH-negative feedback mechanism. As a prohormone, T(4) must be converted to T(3) to acquire biological activity; thus, type 2 iodothyronine deiodinase (D2) is expected to play a critical role in this feedback mechanism. However, the mechanistic details of this pathway are still missing because, counterintuitively, D2 activity is rapidly lost in the presence of T(4) by a ubiquitin-proteasomal mechanism. In the present study, we demonstrate that D2 and TSH are coexpressed in rat pituitary thyrotrophs and that hypothyroidism increases D2 expression in these cells. Studies using two murine-derived thyrotroph cells, TtT-97 and TalphaT1, demonstrate high expression of D2 in thyrotrophs and confirm its sensitivity to negative regulation by T(4)-induced proteasomal degradation of this enzyme. Despite this, expression of the Dio2 gene in TalphaT1 cells is higher than their T(4)-induced D2 ubiquitinating capacity. As a result, D2 activity and net T(3) production in these cells are sustained, even at free T(4) concentrations that are severalfold above the physiological range. In this system, free T(4) concentrations and net D2-mediated T(3) production correlated negatively with TSHbeta gene expression. These results resolve the apparent paradox between the homeostatic regulation of D2 and its role in mediating the critical mechanism by which T(4) triggers the TSH-negative feedback.
Assuntos
Regulação da Expressão Gênica , Iodeto Peroxidase/genética , Hipófise/metabolismo , Tireotropina/fisiologia , Tiroxina/fisiologia , Animais , Linhagem Celular Tumoral , Células Cultivadas , Retroalimentação Fisiológica , Imuno-Histoquímica , Hibridização In Situ , Iodeto Peroxidase/análise , Masculino , Ratos , Ratos Sprague-Dawley , Tireotropina/análise , Tireotropina/genética , Tri-Iodotironina/biossíntese , Iodotironina Desiodinase Tipo IIRESUMO
Mice with one thyroid hormone receptor (TR) alpha-1 allele encoding a dominant negative mutant receptor (TR alpha1(PV/+)) have persistently elevated serum T3 levels (1.9-fold above normal). They also have markedly increased hepatic type 1 iodothyronine deiodinase (D1) mRNA and enzyme activity (4- to 5-fold), whereas other hepatic T3-responsive genes, such as Spot14 and mitochondrial alpha-glycerol phosphate dehydrogenase (alpha-GPD), are only 0.7-fold and 1.7-fold that of wild-type littermates (TR alpha1+/+). To determine the cause of the disproportionate elevation of D1, TR alpha1+/+ and TR alpha1(PV/+) mice were rendered hypothyroid and then treated with T3. Hypothyroidism decreased hepatic D1, Spot14, and alpha-GPD mRNA to similar levels in TR alpha1+/+ and TR alpha1(PV/+) mice, whereas T3 administration caused an approximately 175-fold elevation of D1 mRNA but only a 3- to 6-fold increases in Spot14 and alpha-GPD mRNAs. Interestingly, the hypothyroidism-induced increase in cerebrocortical type 2 iodothyronine deiodinase activity was 3 times greater in the TR alpha1(PV/+) mice, and these mice had no T3-dependent induction of type 3 iodothyronine deiodinase. Thus, the marked responsiveness of hepatic D1 to T3 relative to other genes, such as Spot14 and alpha-GPD, explains the relatively large effect of the modest increase in serum T3 in the TR alpha1(PV/+) mice, and TR alpha plays a key role in T3-dependent positive and negative regulation of the deiodinases in the cerebral cortex.
Assuntos
Biomarcadores , Iodeto Peroxidase/biossíntese , Iodeto Peroxidase/química , Glândula Tireoide/metabolismo , Animais , Córtex Cerebral/metabolismo , Heterozigoto , Hipotireoidismo , Rim/metabolismo , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Biológicos , Mutação , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tiroxina/metabolismo , Fatores de Tempo , Tri-Iodotironina/metabolismoRESUMO
Thyroid hormone affects multiple aspects of bone metabolism, but little is known about thyroid hormone deiodination in bone cells except that cultures of skeletal cells and bone organ express types 1 and 2 iodothyronine deiodinases (D1 and D2) mRNAs. In the present study, outer ring deiodination (ORD) activity was detected in bone extracts of multiple sites of the mouse skeleton, bone marrow, and the MC3T3-E1 osteoblastic cell line. In all tissues, ORD was detected using 125I-rT3 or 125I-T4 as substrates and was found to be 6-n-propylthiouracil insensitive, display a Michaelis constant (T4) of approximately 1 nM, increase about 3-fold in hypo- and virtually disappear in thyrotoxicosis. Extracts of calvaria had the lowest ORD activity, whereas tibial and femoral extracts had roughly three times as much. The absence of ORD activity in bone extracts from mice with targeted disruption of the Dio2 gene confirms the principal role of D2 in this tissue. In the MC3T3-E1 osteoblasts, D2 activity increased in a time-dependent manner after plating, and with the content of selenium in the media, reaching a maximum 5-7 d later as cells attained more than 90% confluence. In these cells D2 half-life is about 30-40 min, which is further accelerated by exposure to substrate and stabilized by the proteasome inhibitor, MG132. Treatment with vitamin D [1,25(OH)2VD]-induced D2 activity by 2- to 3-fold as early as 24 h, regardless of the level of cell confluence, but estradiol, PTH, forskolin, leptin, TNFalpha, TGFbeta, and dexamethasone did not affect D2. Given the role of D2 in other cell types and processes, it is likely that bone ORD not only plays a role in bone development and adult bone T3 homeostasis but also contributes to extrathyroidal T3 production and maintenance of serum T3.
Assuntos
Osso e Ossos/enzimologia , Iodeto Peroxidase/metabolismo , Osteoblastos/citologia , Osteoblastos/enzimologia , Vitamina D/análogos & derivados , Animais , Diferenciação Celular/fisiologia , Linhagem Celular , Meia-Vida , Iodeto Peroxidase/deficiência , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Osteoblastos/efeitos dos fármacos , Fatores de Tempo , Vitamina D/farmacologia , Iodotironina Desiodinase Tipo IIRESUMO
The single-nucleotide polymorphism A/G in the type 2 deiodinase (D2) gene predicts a threonine (Thr) to alanine (Ala) substitution at codon 92 (D2 Thr92Ala) and is associated with insulin resistance in obese patients. Here, this association was investigated in 183 patients with type 2 diabetes mellitus, using homeostasis model assessment. The median fasting plasma insulin in Ala/Ala individuals was significantly higher than in patients with Ala/Thr or Thr/Thr genotypes (19.6 vs. 12.0 vs. 14.8 mIU/ml, respectively; P = 0.004). Assuming a recessive model, the homeostasis model assessment index was higher in the Ala/Ala group when compared with Ala/Thr-Thr/Thr group (8.50 vs. 4.85, P = 0.003). Although this polymorphism has not been associated with changes in D2 kinetics as measured in HEK-293 cells transiently expressing D2 Thr92Ala, we investigated whether such association could be detected in human tissue samples. Remarkably, in thyroid and skeletal muscle samples from subjects homozygous for the Ala allele, D2 velocity was significantly lower than in subjects with Ala/Thr-Thr/Thr genotypes (P = 0.05 and 0.04, respectively). In conclusion, the A/G polymorphism is associated with greater insulin resistance in type 2 diabetes mellitus patients and with lower D2 velocity in tissue samples. These findings suggest that the D2-generated T(3) in skeletal muscle plays a role in insulin resistance.
Assuntos
Substituição de Aminoácidos , Diabetes Mellitus Tipo 2/genética , Resistência à Insulina/genética , Iodeto Peroxidase/genética , Iodeto Peroxidase/metabolismo , Polimorfismo de Nucleotídeo Único , Alanina , Humanos , Cinética , Treonina , Iodotironina Desiodinase Tipo IIRESUMO
Type 2 deiodinase (D2) is a selenoenzyme catalyzing the activation of T(4) to T(3). D2 activity/mRNA ratios are often low, suggesting that there is significant posttranscriptional regulation. The D2 mRNA in higher vertebrates is more than 6 kb, containing long 5' and 3' untranslated regions (UTRs). The D2 5'UTRs are greater than 600 nucleotides and contain 3-5 short open reading frames. These full-length 5'UTRs reduce the D2 translation efficiency approximately 5-fold. The inhibition by human D2 5'UTR is localized to a region containing the first short open reading frame encoding a tripeptide-MKG. This inhibition was abolished by mutating the AUG start codon and weakened by modification of the essential purine of the Kozak consensus. Deletion of the 3.7-kb 3'UTR of the chicken D2 mRNA increased D2 activity approximately 3.8-fold due to an increase in D2 mRNA half-life. In addition, alternatively spliced D2 mRNA transcripts similar in size to the major 6- to 7-kb D2 mRNAs but not encoding an active enzyme are present in both human and chicken tissues. Our results indicate that a number of factors reduce the D2 protein levels. These mechanisms, together with the short half-life of the protein, ensure limited expression of this key regulator of T(4) activation.
Assuntos
Processamento Alternativo , Iodeto Peroxidase/genética , Processamento Pós-Transcricional do RNA , RNA Mensageiro/química , Regiões 3' não Traduzidas , Regiões 5' não Traduzidas , Sequência de Aminoácidos , Animais , Sequência de Bases , Células Cultivadas , Galinhas/genética , Clonagem Molecular , Códon de Iniciação , Elementos de DNA Transponíveis , Regulação Enzimológica da Expressão Gênica , Meia-Vida , Humanos , Iodeto Peroxidase/metabolismo , Dados de Sequência Molecular , Mutação , Conformação de Ácido Nucleico , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , Iodotironina Desiodinase Tipo IIRESUMO
The type 2 monodeiodinase (D2) is an endoplasmic reticulum-resident membrane selenoprotein responsible for catalyzing the first step in thyroid hormone action, T(4) deiodination to T(3). Its short half-life is due to ubiquitination and proteolysis by proteasomes, a mechanism that is accelerated by D2 interaction with T(4). To identify proteins involved in D2 ubiquitination, a FLAG-tagged selenocystine133-to-Cys mutation of the human D2 (CysD2) was created and expressed in Saccharomyces cerevisiae using the GAL1 gene promoter. CysD2 activity was detected in the microsomes, indistinguishable from transiently expressed CysD2 in vertebrate cells. Treatment with 100 mg/ml cycloheximide or 30 micro M T(4) caused rapid loss of CysD2 (t(1/2) = approximately 30 min). Clasto-lactacystin beta-lactone not only increased galactose-inducible CysD2 but also stabilized CysD2 in the presence of cycloheximide or T(4). Immunoprecipitation with anti-FLAG antibody combined with Western analysis with antiubiquitin revealed that CysD2 is heavily ubiquitinated. Expression of CysD2 in yeast strains that lack the ubiquitin conjugases Ubc6p or Ubc7p stabilized CysD2 half-life by markedly reducing CysD2 ubiquitination, whereas no difference was detected in Ubc1p-deficient mutants. Similarly, expression of CysD2 in UBC6 and UBC7 mutants also impaired the substrate-induced loss of CysD2 activity and protein. In conclusion, Ubc6p and Ubc7p are required for normal and substrate-induced ubiquitination and proteolysis of D2.
Assuntos
Retículo Endoplasmático/metabolismo , Iodeto Peroxidase/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Western Blotting , Linhagem Celular , Cisteína Endopeptidases/metabolismo , Retículo Endoplasmático/enzimologia , Galactose/metabolismo , Deleção de Genes , Meia-Vida , Humanos , Iodeto Peroxidase/genética , Cinética , Complexos Multienzimáticos/metabolismo , Complexo de Endopeptidases do Proteassoma , Ligação Proteica , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Tiroxina/metabolismo , Tri-Iodotironina/metabolismo , Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/genética , Iodotironina Desiodinase Tipo IIRESUMO
Whereas many cardiac symptoms of thyrotoxicosis resemble those of the hyperadrenergic state, circulating catecholamines are reduced or normal in this condition. To test the hypothesis that the thyrotoxic heart is hypersensitive to catechol-amines, we studied beta-adrenergic signaling in a transgenic (TG) mouse in which the human type 2 iodothyronine deiodinase (D2) gene is expressed in myocardium. Because D2 converts T4 to T3, the active form of thyroid hormone, the D2 TG mouse exhibits mild, chronic thyrotoxicosis that is limited to the myocardium. In the current study, we determined that cAMP accumulation in response to either norepinephrine or forskolin treatment was increased in isolated ventricular myocardiocytes and membrane-enriched fractions prepared from these D2 TG hearts as compared with wild type. This increase in adenylyl cyclase (AC) Vmax could not be explained by changes in AC isoform expression or changes in the long or short forms of stimulatory G-protein Gsalpha, which were approximately 10% decreased in D2 TG membranes. However, Western analysis and ADP-ribosylation studies suggest that the increase in AC Vmax is mediated by a decrease in the expression of inhibitory G proteins (Gialpha-3 and/or Goalpha). These data suggest that cardiac thyrotoxicosis leads to increased beta-adrenergic responsiveness of cardiomyocytes via alterations in the regulatory G-protein elements of the AC membrane complex.
Assuntos
Iodeto Peroxidase/genética , Miocárdio/metabolismo , Receptores Adrenérgicos beta/metabolismo , Tireotoxicose/metabolismo , Adenilil Ciclases/metabolismo , Animais , Membrana Celular/metabolismo , Células Cultivadas , Colforsina/farmacologia , AMP Cíclico/metabolismo , Feminino , Proteínas de Ligação ao GTP/efeitos dos fármacos , Proteínas de Ligação ao GTP/metabolismo , Coração/efeitos dos fármacos , Ventrículos do Coração/metabolismo , Iodeto Peroxidase/metabolismo , Isoenzimas/metabolismo , Cinética , Masculino , Camundongos , Camundongos Transgênicos , Miocárdio/patologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Norepinefrina/farmacologia , Tireotoxicose/fisiopatologia , Iodotironina Desiodinase Tipo IIRESUMO
The type 2 iodothyronine selenodeiodinase (D2) is an endoplasmic reticulum (ER)-resident selenoprotein that activates T4 to T3, playing a critical role in thyroid homeostasis. D2 has an approximately 45-min half-life due to selective ubiquitin-mediated ER-associated degradation (ERAD), a process of particular interest because it is accelerated by exposure to D2 substrates, T4 or rT3. The present in vitro binding studies indicate that glutathione-S-transferase (GST)-human D2 fusion proteins specifically associate with a mammalian homolog of the ubiquitin conjugase UBC7 (MmUBC7), with localization to amino acids 169-234 of D2. Coexpression of D2 with an inactive D2 mutant or a truncated version containing amino acids 169-234 stabilizes D2 half-life, supporting the importance of the carboxyl region of D2 for ERAD. Mammalian UBC6 (MmUBC6) does not directly associate with D2 but can associate with a complex containing UBC7 and D2. At the same time, functional studies in human embryonic kidney-293 cells indicate that D2 activity half-life and protein levels are stabilized only when inactive mutants of both UBC6 and UBC7 are overexpressed with D2, suggesting that redundancy may exist at the level of the E2 for both basal and substrate-accelerated D2 ERAD. In conclusion, D2 ERAD in human cells proceeds via an association between UBC7 and the carboxyl region of D2, a unique mechanism for the control of thyroid hormone activation.