Comparison of the physicochemical properties of type I and type II iodothyronine 5\u27-deiodinase

The 5\u27-deiodination of thyroxine is catalyzed by two enzymes which differ in their tissue distribution, substrate specificities, sensitivity to the inhibitor, propylthiouracil, and response to thyroid status. By using the affinity label, N-bromoacetyl-L-thyroxine, both isoenzymes have been found to have substrate binding subunits of approximately 27 kDa. In this study, we compared the substrate binding subunits and hydrodynamic properties of the type I and the type II isozymes using the affinity label, N-bromoacetyl-L-thyroxine, to identify the enzymes. High resolution sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the substrate binding subunit of the type I enzyme had an Mr of 27,000, while that of the type II enzyme had a slightly higher Mr of 29,000. This difference was not accounted for by glycosylation. Partial staphylococcal V8-protease digests of the substrate binding subunit of the type I enzyme yielded fragments of 14.6, 13.7, and 7.0 kDa, while V8-protease digests of the substrate binding subunit for the type II enzyme produced fragments of 28.0, 25.1, 19.0, 9.5, 7.2, and 5.8 kDa. Unique cyanogen bromide fragmentation patterns were also observed for the two substrate binding subunits. Sedimentation coefficients of the detergent-soluble type I and type II holoenzymes were 3.67 and 5.22 S, respectively, as determined by sucrose density centrifugation. The type I enzyme behaved as a globular protein, whereas the type II enzyme showed sedimentation properties typical of asymmetric integral membrane proteins. The Stokes radii were 3.78 and 4.97 nm, respectively. From these data, the calculated Mr for detergent-solubilized type I 5\u27-iodothyronine deiodinase was 55,400 and for the type II enzyme was 198,700. These data indicate that the two isozymes of iodothyronine 5\u27-deiodinase are multimeric, differ in holoenzyme size and subunit composition, and that their substrate binding subunits are distinct

Thyroid hormone-dependent redistribution of the 55-kilodalton monomer of protein disulfide isomerase in cultured glial cells

In addition to the effects of thyroid hormone that are mediated through interaction with chromatin-associated receptors, T4 modulates the activity of the cellular content of the membrane-associated protein type II iodothyronine 5\u27-deiodinase (5\u27D-II) by regulating its degradation through an actin-dependent extranuclear mechanism. Under the influence of thyroid hormone, the substrate-binding subunit of 5\u27D-II is translocated from the plasma membrane to an intracellular microfilament-associated pool. In glial cells, a 55-kilodalton (kDa) protein (glial-p55), which was shown to be identical to the 55-kDa monomer of protein disulfide isomerase (PDI) also demonstrates a similar T4-dependent association to the F-actin microfilaments. To explore the role of glial-p55 in the extranuclear effect of thyroid hormone in glial cells, the effects of thyroid hormone on the subcellular localization of glial-p55 were further examined. The current study demonstrates the presence of two pools of glial-p55. While the majority of glial-p55 is associated with endoplasmic reticulum and represents PDI, approximately 25% of glial-p55 is cytosolic in the absence of thyroid hormone. Cytosolic glial-p55 is lost from the cells after mild permeabilization with saponin, and treatment of cells with T4 causes the shift of glial-p55 from the cytosolic pool to the subcellular fractions that contain the actin cytoskeleton. Crude microsomal preparations were prepared which contain membranes, microfilaments, and other particulate cell structures. In the absence of thyroid hormone, glial cells lack an intact actin cytoskeleton, and glial-p55 is easily removed from these preparations by conditions that remove extrinsic membrane proteins like PDI, such as alkaline pH and detergent extraction. In contrast, glial-p55 is not removed from the crude microsomes prepared from thyroid hormone-replete glial cells that contain an intact actin cytoskeleton. Since previous work in our laboratory indicated that glial-p55 becomes actin associated in a thyroid-dependent manner along with the substrate-binding subunit of 5\u27D-II, this study suggests that the 55-kDa monomer of PDI may play a role in the thyroid hormone-dependent regulation of actin polymerization and the degradation of 5\u27D-II

Catalytic activity of type II iodothyronine 5\u27-deiodinase polypeptide is dependent upon a cyclic AMP activation factor

Type II iodothyronine 5\u27-deiodinase is an approximately 200-kDa multimeric enzyme in the brain that catalyzes the deiodination of thyroxine (T4) to its active metabolite, 3,5,3\u27-triiodothyronine. In astrocytes, cAMP stimulation is required to express catalytically active type II iodothyronine 5\u27-deiodinase. The affinity ligand N-bromoacetyl-L-T4 specifically labels the 29-kDa substrate-binding subunit (p29) of this enzyme in cAMP-stimulated astrocytes. To determine the requirements for cAMP-induced activation of this enzyme, we optimized N-bromoacetyl-L-T4 labeling of p29 in astrocytes lacking type II iodothyronine 5\u27-deiodinase activity and examined the effects of cAMP on the hydrodynamic properties and subcellular location of the enzyme. We show that the p29 subunit is expressed in unstimulated astrocytes and requires 10-fold higher concentrations of N-bromoacetyl-L-T4 to achieve incorporation levels equal to those of p29 in cAMP-stimulated cells. Gel filtration showed that p29 was part of a multimeric membrane-associated complex in both cAMP-stimulated and unstimulated astrocytes and that cAMP stimulation led to an increase of approximately 60 kDa in the mass of the holoenzyme. In unstimulated astrocytes, p29 resides in the perinuclear space. Cyclic AMP stimulation leads to the translocation of p29 to the plasma membrane coincident with the appearance of deiodinating activity. These data show that cAMP-dependent activation of type II iodothyronine 5\u27-deiodinase activity results from the synthesis of additional activating factor(s) that associates with inactive enzyme and leads to the translocation of enzyme polypeptide(s) from the perinuclear space to the plasma membrane

Structural requirements of iodothyronines for the rapid inactivation and internalization of type II iodothyronine 5\u27-deiodinase in glial cells

3,3\u275,5\u27-Tetraiodothyronine (T4), but not 3,3\u275-triiodothyronine (T3), acutely regulates the activity of the plasma membrane-bound enzyme, type II iodothyronine 5\u27-deiodinase (5\u27D-II), by inducing internalization of the enzyme through an extranuclear, energy-dependent mechanism that requires an intact actin cytoskeleton. The affinity label, N-bromoacetyl-L-T4, binds to 5\u27D-II and irreversibly inhibits the enzyme but does not initiate internalization. To determine the structural elements of T4 which are required for enzyme internalization, T4 analogs were modified in the alanine side chain and were then evaluated for their ability to induce enzyme internalization, to inhibit enzyme activity, and to promote actin polymerization in hypothyroid cells. The analogs studied showed marked variability in their ability to inactivate 5\u27D-II. The rank order of potency for enzyme inactivation was T4 \u3e COOH-blocked analogs \u3e NH3 and COOH blocked analogs \u3e\u3e NH3 blocked analogs (EC50 values range from 1 to \u3e 1000 nM). In contrast, all T4 analogs tested and T4 were excellent competitive inhibitors of 5\u27D-II with respect to substrate (Ki values ranged from 4 to 27 nM). The differential capability of iodothyronines to inactivate the enzyme was not related to their ability to enter the cell, since Ki values measured in intact glial cells were equivalent to those measured in cell sonicates. The power of the T4 analogs to inactivate 5\u27D-II was paralleled by their ability to polymerize actin in hypothyroid cells and to induce 5\u27D-II binding to F-actin. The data show that modification of the alanine side chain markedly alters the ability of T4 analogs to induce 5\u27D-II inactivation and actin polymerization. A net negative charge on the alanine side chain of T4 is detrimental for the hormone-dependent inactivation of 5\u27D-II and polymerization of actin, whereas uncharged or positively charged molecules retain significant activity

Degradation and recycling of the substrate-binding subunit of type II iodothyronine 5\u27-deiodinase in astrocytes

Thyroxine dynamically regulates levels of type II iodothyronine 5\u27-deiodinase (5\u27D-II) by modulating enzyme inactivation and targeting the enzyme to different pathways of internalization. 5\u27D-II is an approximately 200-kDa multimeric protein containing a 29-kDa substrate-binding subunit (p29) and an unknown number of other subunits. In the absence of thyroxine (T4), p29 is slowly endocytosed and transported to the lysosomes. T4 treatment rapidly activates an actin-mediated endocytotic pathway and targets the enzyme to the endosomes. In this study, we have characterized the influence of T4 on the intracellular trafficking of 5\u27D-II. We show that T4 accelerates the rate of 5\u27D-II inactivation by translocating the enzyme to the interior of the cell and by sequestering p29 in the endosomal pool without accelerating the rate of degradation of p29. This dichotomy between the rapid inactivation of catalytic activity and the much slower degradation of p29 is consistent with the reuse of p29 in the production of 5\u27D-II activity. Immunocytochemical analysis with a specific anti-p29 IgG shows that pulse affinity-labeled p29 reappears on the plasma membrane approximately 2 h after enzyme internalization in the presence of T4, indicating that p29 is recycled. Despite the ability of p29 to be recycled in the T4-treated cell, 5\u27D-II catalytic activity requires ongoing protein synthesis, presumably of another enzyme component(s) or an accessory enzyme-related protein. In the absence of T4, enzyme inactivation and p29 degradation are temporally linked, and pulse affinity-labeled p29 is internalized and sequestered in discrete intracellular pools. These data suggest that T4 regulates fundamental processes involved with the turnover of integral membrane proteins and participates in regulating the inter-relationships between the degradation, recycling, and synthetic pathways

The role of cell cycle regulatory protein, cyclin D1, in the progression of thyroid cancer

Cell cycle progression is facilitated by cyclin-dependent kinases that are activated by cyclins including cyclin D1 and inactivated by cyclin-dependent kinase inhibitors (CDKIs) such as p27. Our previous studies have demonstrated decreased p27 expression in both papillary and more aggressive carcinomas of the thyroid compared to thyroid adenoma and almost similar level of cyclin D1 expression between thyroid adenoma and papillary carcinoma. These results indicate that CDKIs may have an important role in the carcinogenesis of the thyroid and that they probably have a limited role in malignant progression of the thyroid cancer. The role of cyclin D1 in malignant progression of thyroid carcinoma has yet to be established. We studied the expression of cyclin D1 by immunohistochemistry in 34 cases of conventional papillary carcinoma (CPC), 10 cases of minimally invasive follicular carcinoma (MIFC), and 32 cases of more aggressive thyroid carcinoma (ATC), which included 11 tall cell variants, one columnar cell variant of papillary carcinoma, seven insular carcinomas, and 13 anaplastic carcinomas. Cyclin D1 staining was classified by staining score as 0, negative; 1+, less than 25%; 2+, 25 to 50%; and 3+, more than 50% tumor cells staining positive. Kruskal-Wallis one-way ANOVA and Wilcoxon Rank Sum/Mann-Whitney U Test was used to assess the difference in the expression of cyclin D1 between the study groups. Twenty-eight out of the 34 CPCs were cyclin D1 positive, 24 (70%) were 1+, 3 (9%) were 2+, and one (3%) were 3+ positive. Seven of 10 MIFCs were cyclin D1 positive, five (71%) were 1+, and the remaining two (29%) were 2+ positive. On the other hand, 28 of 32 ATCs showed cyclin D1 immunostaining. Of these, three (9%) were 1+, five (13%) were 2+, and 20 (63%) were 3+ positive. This study demonstrates a significant overexpression of cyclin D1 in ATC compared CPC (P \u3c .001) and MIFC (P \u3c .005), suggesting that the cyclin D1 expression may play a role in tumor progression and may have prognostic significance in thyroid cancer

The mammalian homolog of the frog type II selenodeiodinase does not encode a functional enzyme in the rat

Type II iodothyronine deiodinase is a short-lived, membrane-bound enzyme found in rat brain, brown adipose tissue, and cAMP-stimulated astrocytes. Recently, a full-length complementary DNA (cDNA) encoding a 30-kDa, type II-like selenodeiodinase was cloned from frog, and a homologous partial cDNA (rBAT 1.1), containing two in-frame selenocysteine codons (UGA), was isolated from rat brown adipose tissue. Importantly, the rBAT 1.1 cDNA was derived from a 7.5-kb messenger RNA (mRNA) and did not encode a functional selenoenzyne unless an enabling selenocysteine insertion sequence was appended to the presumed coding region and this cDNA. In this study we determined whether the native 7.5-kb SeD2 mRNA in rat tissues programmed the synthesis of the native type II deiodinase using specific antibodies that were raised against the C-terminus of full-length, 30-kDa SeD2 protein and against the catalytic core of SeD2. Direct analysis of the translation products programmed by the native SeD2 mRNA in cAMP-stimulated astrocytes was performed using antisense deoxynucleotides and hybrid selection strategies. (Bu)2cAMP-stimulated rat astrocytes expressed both type II deiodinase activity (approximately 2500 U/mg protein) and contained abundant levels of the 7.5-kb SeD2 mRNA. However, no immunoreactive 30-kDa SeD2 protein was identified by Western analysis, immunoprecipitation, or immunocytochemistry, and the specific C-terminus antiserum failed to immunoprecipitate deiodinase activity from (Bu)2cAMP-stimulated astrocytes, brown adipose tissue or brain. Instead, the native 7.5-kb SeD2 mRNA encoded a 15-kDa protein that terminated at the first UGA codon and contained the catalytically inactive, N-terminal 129 amino acids of SeD2. These data show that the native 7.5-kb SeD2 mRNA in stimulated astrocytes does not encode D2

Cloning, expression, and functional characterization of the substrate binding subunit of rat type II iodothyronine 5\u27-deiodinase

Type II iodothyronine 5\u27-deiodinase catalyzes the bioactivation of thyroid hormone in the brain. In astrocytes, this approximately 200-kDa, membrane-bound enzyme is composed of at least one p29 subunit, an approximately 60-kDa, cAMP-induced activation protein, and one or more unidentified catalytic subunit(s). Recently, an artificial type II-like selenodeiodinase was engineered by fusing two independent cDNAs together; however, no native type II selenodeiodinase polypeptide is translated in the brain or brown adipose tissue of rats. These data suggest that the native type II 5\u27-deiodinase in rat brain is unrelated to this artificial selenoprotein. In this report, we describe the cloning of the 29-kDa subunit (p29) of type II 5\u27-deiodinase from a lambdazapII cDNA library prepared from cAMP-induced astrocytes. The 3.3-kilobase (kb) cDNA encodes an approximately 30-kDa, 277-amino acid long, hydrophobic protein lacking selenocysteine. Northern blot analysis showed that a 3.5-kb p29 mRNA was present in tissues showing type II 5\u27-deiodinase activity such as brain and cAMP-stimulated astrocytes. Domain-specific, anti-p29 antibodies specifically immunoprecipitated enzyme activity. Overexpression of exogenous p29 or a green fluorescence protein (GFP)-tagged p29 fusion protein led to a \u3e100-fold increase in deiodinating activity in cAMP-stimulated astrocytes, and the increased activity was specifically immunoprecipitated by anti-GFP antibodies. Steady-state reaction kinetics of the enzyme in GFP-tagged p29-expressing astrocytes are identical to those of the native enzyme in brain. Direct injection of replication-deficient Ad5-p29(GFP) virus particles into the cerebral cortex of neonatal rats leads to a approximately 2-fold increase in brain type II 5\u27-deiodinating activity. These data show 1) that the 3.3-kb p29 cDNA encodes an essential subunit of rat type II iodothyronine 5\u27-deiodinase and 2) identify the first non-selenocysteine containing subunit of the deiodinase family of enzymes

Politics in Europe [6th ed.]

No abstract available

Effects of selenium deficiency on thyroid hormone economy in rats

In selenium-deficient rats, peripheral T4 to T3 conversion is markedly decreased due to the loss of the selenoprotein, type I iodothyronine 5\u27-deiodinase (5\u27D-I). Despite the marked increase in circulating T4 that results from this loss of 5\u27D-I, serum T3 concentrations in selenium-deficient rats remain in the normal range. To determine the physiological mechanism(s) that maintains circulating T3 when peripheral T4 to T3 conversion is impaired, we examined the interrelationships between selenium intake and the metabolism of T3 and T4 in the rat. In euthyroid rats, selenium deficiency caused the expected loss of 5\u27D-I, with a 52% increase in serum T4, which paralleled an increase in the T4 biological half-life. Consistent with the prolonged t1/2 of T4, short term thyroidectomy (48 h) in selenium-deficient rats failed to decrease serum T4 concentrations to the levels observed in short term thyroidectomized, selenium-supplemented rats. Short term thyroidectomy also caused an expected 33% decrease in liver 5\u27D-I and a 44% increase in brain type II iodothyronine 5\u27-deiodinase (5\u27D-II) activities in selenium-supplemented rats. However, in selenium-deficient rats, short term thyroidectomy did not affect 5\u27D-I or 5\u27D-II activities. In contrast to the selenium-dependent changes in circulating T4 levels, little or no change in circulating T3 concentrations occurred. There was a 20% increase in the T3 half-life in selenium-deficient rats. The serum T3 sulfate concentration was increased, and T3 deiodination was reciprocally decreased in the selenium-deficient rats. These data suggest that increased T3 sulfate generation in selenium-deficient rats may lead to greater T3 availability through enterohepatic recycling of the iodothyronine and may explain why there are only minor changes in serum T3 concentrations in selenium-deficient rats