RESUMO
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
For T(3) to mediate its biological effects, the prohormone T(4) must be activated by removal of an outer-ring iodine by the type 1 or 2 deiodinases (D1 and D2) with approximately 60% of the daily T(3) production in rodents being produced extrathyroidally through this pathway. To further define the role of these enzymes in thyroid hormone homeostasis, we backcrossed the targeted disruption of the Dio2 gene into C3H/HeJ (C3H) mice with genetically low D1 expression to create the C3H-D2KO mouse. Remarkably, these mice maintain euthyroid serum T(3) levels with normal growth and no decrease in expression of hepatic T(3)-responsive genes. However, serum T(4) is increased 1.2-fold relative to the already elevated C3H levels, and serum TSH is increased 1.4-fold. Despite these increases, thyroidal (125)I uptake indicates no difference in thyroidal activity between C3H-D2KO and C3H mice. Although C3H-D2KO hepatic and renal D1 activities were well below those observed in wild-type mice (approximately 0.1-fold for both), they were 8-fold and 2-fold higher, respectively, relative to C3H mice. Thyroidal D1 and cerebral cortical type 3 deiodinase activity were unchanged between C3H-D2KO and C3H mice. In conclusion, C3H-D2KO mice have notably elevated serum T(4) levels, and this, in conjunction with residual D1 activity, is likely an important role in the maintenance of euthyroid serum T(3) concentrations.
Assuntos
Iodeto Peroxidase/genética , Tiroxina/metabolismo , Tri-Iodotironina/sangue , Tri-Iodotironina/metabolismo , Animais , Cruzamentos Genéticos , Feminino , Iodeto Peroxidase/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Knockout , Iodotironina Desiodinase Tipo IIRESUMO
Thyroid hormone activation is catalyzed by two deiodinases, D1 and D2. Whereas D1 is a stable plasma membrane protein, D2 is resident in the endoplasmic reticulum (ER) and has a 20-min half-life due to selective ubiquitination and proteasomal degradation. Here we have shown that stable retention explains D2 residency in the ER, a mechanism that is nevertheless over-ridden by fusion to the long-lived plasma membrane protein, sodium-iodine symporter. Fusion to D2, but not D1, dramatically shortened sodium-iodine symporter half-life through a mechanism dependent on an 18-amino acid D2-specific instability loop. Similarly, the D2-specific loop-mediated protein destabilization was also observed after D2, but not D1, was fused to the stable ER resident protein SEC62. This indicates that the instability loop in D2, but not its subcellular localization, is the key determinant of D2 susceptibility to ubiquitination and rapid turnover rate. Our data also show that the 6 N-terminal amino acids, but not the 12 C-terminal ones, are the ones required for D2 recognition by WSB-1.
Assuntos
Iodeto Peroxidase/metabolismo , Sequência de Aminoácidos , Domínio Catalítico , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Glicosilação , Humanos , Iodeto Peroxidase/química , Proteínas de Membrana Transportadoras/química , Dados de Sequência Molecular , Mutação , Conformação Proteica , Estrutura Terciária de Proteína , Simportadores/química , Iodotironina Desiodinase Tipo IIRESUMO
Somatostatin (somatotropin release inhibitory factor; SRIF) is an endogenous peptide produced at sites of inflammation, making the SRIF a candidate in regulating vascular inflammation. We have used primary human coronary artery endothelial cells (hCAEC) as a model to study SRIF's vascular actions. RT-PCR analysis of hCAEC total mRNA demonstrated the presence of the sst(4) receptor subtype, providing a target for SRIF intracellular signaling. Western blotting with phospho-specific ERK1/2 antibodies showed that SRIF-14 acutely inhibited basal phosphorylation of the extracellular regulated kinases (ERK1/2) by 80%. In addition, SRIF-14 treated hCAEC cell lysates showed a 2.6-fold increase in phosphatase activity, which was inhibited by sodium vanadate. Furthermore, SRIF-14 appeared to be anti-inflammatory in hCAEC as IL-1beta-induced adhesion molecule expression was reduced by 50%. Together, these results show that the coronary artery endothelium is a direct target of SRIF action.