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1.
J Physiol Pharmacol ; 65(2): 257-72, 2014 Apr.
Article in English | MEDLINE | ID: mdl-24781735

ABSTRACT

The present study examined human postmortem brains for changes consistent with the hypothesis of local brain TH deficiency in autism spectrum disorders (ASD). Brain levels of oxidative stress marker - 3-nitrotyrosine (3-NT), iodothyronine deiodinase type 2(D2) and type 3 (D3), 3',3,5-triiodothyronine (T3) content, mercury content and gene expression levels were analyzed and compared in the several regions of postmortem brains derived from both male and female control and ASD cases, age 4-16 years. We report that some parameters measured, such as D2 are subject to rapid postmortem inactivation, while others that were analyzed showed both brain region- and sex-dependent changes. Levels of 3-NT were overall increased, T3 was decreased in the cortical regions of ASD brains, while mercury levels measured only in the extracortical regions were not different. The expression of several thyroid hormone (TH)-dependent genes was altered in ASD. Data reported here suggest the possibility of brain region-specific disruption of TH homeostasis and gene expression in autism.


Subject(s)
Brain/metabolism , Child Development Disorders, Pervasive/genetics , Child Development Disorders, Pervasive/metabolism , Thyroid Hormones/metabolism , Adolescent , Animals , Child , Child, Preschool , Female , Gene Expression , Homeostasis , Humans , Iodide Peroxidase/metabolism , Male , Mercury/metabolism , Rats, Sprague-Dawley , Triiodothyronine, Reverse/metabolism , Tyrosine/analogs & derivatives , Tyrosine/metabolism , Iodothyronine Deiodinase Type II
2.
J Endocrinol Invest ; 34(5): 395-407, 2011 May.
Article in English | MEDLINE | ID: mdl-21427525

ABSTRACT

T4 is a prohormone secreted by the thyroid. T4 has a long half life in circulation and it is tightly regulated to remain constant in a variety of circumstances. However, the availability of iodothyronine selenodeiodinases allow both the initiation or the cessation of thyroid hormone action and can result in surprisingly acute changes in the intracellular concentration of the active hormone T3, in a tissue- specific and chronologically-determined fashion, in spite of the constant circulating levels of the prohormone. This fine-tuning of thyroid hormone signaling is becoming widely appreciated in the context of situations where the rapid modifications in intracellular T3 concentrations are necessary for developmental changes or tissue repair. Given the increasing availability of genetic models of deiodinase deficiency, new insights into the role of these important enzymes are being recognized. In this review, we have incorporated new information regarding the special role played by these enzymes into our current knowledge of thyroid physiology, emphasizing the clinical significance of these new insights.


Subject(s)
Iodide Peroxidase/physiology , Animals , Feedback, Physiological , Humans , Molecular Structure , Thyroid Diseases/physiopathology , Thyroid Gland/enzymology , Thyroid Gland/physiology , Thyroxine/chemistry , Thyroxine/metabolism , Triiodothyronine/chemistry , Triiodothyronine/metabolism
3.
J Clin Endocrinol Metab ; 88(2): 594-8, 2003 Feb.
Article in English | MEDLINE | ID: mdl-12574186

ABSTRACT

Thyroid function is normally undisturbed in patients with thyroid carcinoma. We have identified three patients with large or widely metastatic follicular thyroid carcinoma who had a persistently increased ratio of serum T(3) to T(4) in the absence of autonomous production of T(3) by the tumor. To investigate the possibility of tumor-mediated T(4) to T(3) conversion, we assayed types 1 and 2 iodothyronine selenodeiodinase (D1 and D2) activity in a 965-g follicular thyroid carcinoma resected from one of these patients. The V(max) for D2 was 8-fold higher than in normal human thyroid tissue. Resection of this tumor, leaving the left thyroid lobe intact, normalized the serum T(3) to T(4) ratio. In two other patients, treatment with sufficient levothyroxine to suppress TSH was associated with a high normal T(3) and a subnormal free T(4) index. In one, concomitant administration of the D1 inhibitors, propylthiouracil and propranolol, did not decrease the elevated serum T(3) to T(4) ratio. These data illustrate that increased T(4) to T(3) conversion in follicular thyroid carcinomas, probably by D2, can cause a significant perturbation in peripheral thyroid hormone concentrations.


Subject(s)
Adenocarcinoma, Follicular/enzymology , Iodide Peroxidase/metabolism , Thyroid Neoplasms/enzymology , Thyroxine/blood , Adenocarcinoma, Follicular/blood , Adenocarcinoma, Follicular/surgery , Adult , Aged , Humans , Male , Thyroid Neoplasms/blood , Thyroid Neoplasms/surgery , Thyroid Nodule/blood , Thyroid Nodule/enzymology , Thyroid Nodule/surgery , Iodothyronine Deiodinase Type II
4.
J Clin Invest ; 108(9): 1379-85, 2001 Nov.
Article in English | MEDLINE | ID: mdl-11696583

ABSTRACT

Type 2 iodothyronine deiodinase (D2) is a selenoenzyme, the product of the recently cloned cAMP-dependent Dio2 gene, which increases 10- to 50-fold during cold stress only in brown adipose tissue (BAT). Here we report that despite a normal plasma 3,5,3'-triiodothyronine (T3) concentration, cold-exposed mice with targeted disruption of the Dio2 gene (Dio2(-/-)) become hypothermic due to impaired BAT thermogenesis and survive by compensatory shivering with consequent acute weight loss. This occurs despite normal basal mitochondrial uncoupling protein 1 (UCP1) concentration. In Dio2(-/-) brown adipocytes, the acute norepinephrine-, CL316,243-, or forskolin-induced increases in lipolysis, UCP1 mRNA, and O(2) consumption are all reduced due to impaired cAMP generation. These hypothyroid-like abnormalities are completely reversed by a single injection of T3 14 hours earlier. Recent studies suggest that UCP1 is primarily dependent on thyroid hormone receptor beta (TR beta) while the normal sympathetic response of brown adipocytes requires TR alpha. Intracellularly generated T3 may be required to saturate the TR alpha, which has an approximately fourfold lower T3-binding affinity than does TR beta. Thus, D2 is an essential component in the thyroid-sympathetic synergism required for thermal homeostasis in small mammals.


Subject(s)
Adipose Tissue, Brown/physiology , Iodide Peroxidase/chemistry , Iodide Peroxidase/physiology , Adipose Tissue, Brown/metabolism , Animals , Body Weight , Cells, Cultured , Colforsin/pharmacology , Cyclic AMP/metabolism , Dioxoles/pharmacology , Dose-Response Relationship, Drug , Homeostasis , Hypoglycemic Agents/pharmacology , Iodide Peroxidase/genetics , Mice , Mice, Inbred C57BL , Mice, Transgenic , Mitochondria/metabolism , Models, Biological , Oxygen/metabolism , RNA, Messenger/metabolism , Temperature , Time , Time Factors , Triglycerides/metabolism , Triiodothyronine/blood , Weight Loss , Iodothyronine Deiodinase Type II
5.
Biofactors ; 14(1-4): 17-24, 2001.
Article in English | MEDLINE | ID: mdl-11568436

ABSTRACT

The mechanism of selenocysteine incorporation in eukaryotes has been assumed for almost a decade to be inherently different from that in prokaryotes, due to differences in the architecture of selenoprotein mRNAs in the two kingdoms. After extensive efforts in a number of laboratories spanning the same time frame, some of the essential differences between these mechanisms are finally being revealed, through identification of the factors catalyzing cotranslational selenocysteine insertion in eukaryotes. A single factor in prokaryotes recognizes both the selenoprotein mRNA, via sequences in the coding region, and the unique selenocysteyl-tRNA, via both its secondary structure and amino acid. The corresponding functions in eukaryotes are conferred by two distinct but interacting factors, one recognizing the mRNA, via structures in the 3' untranslated region, and the second recognizing the tRNA. Now, with these factors in hand, crucial questions about the mechanistic details and efficiency of this intriguing process can begin to be addressed.


Subject(s)
3' Untranslated Regions/genetics , Peptide Elongation Factors/metabolism , Proteins/genetics , RNA, Transfer, Amino Acid-Specific/metabolism , Selenocysteine/metabolism , 3' Untranslated Regions/metabolism , Animals , Eukaryotic Cells/metabolism , Methanococcus/genetics , Methanococcus/metabolism , Protein Biosynthesis , RNA, Messenger/genetics , RNA-Binding Proteins/metabolism , Selenoproteins
6.
J Mol Biol ; 310(4): 699-707, 2001 Jul 20.
Article in English | MEDLINE | ID: mdl-11453681

ABSTRACT

Termination of translation in eukaryotes is catalyzed by eRF1, the stop codon recognition factor, and eRF3, an eRF1 and ribosome-dependent GTPase. In selenoprotein mRNAs, UGA codons, which typically specify termination, serve an alternate function as sense codons. Selenocysteine incorporation involves a unique tRNA with an anticodon complementary to UGA, a unique elongation factor specific for this tRNA, and cis-acting secondary structures in selenoprotein mRNAs, termed SECIS elements. To gain insight into the interplay between the selenocysteine insertion and termination machinery, we investigated the effects of overexpressing eRF1 and eRF3, and of altering UGA codon context, on the efficiency of selenoprotein synthesis in a transient transfection system. Overexpression of eRF1 does not increase termination at naturally occurring selenocysteine codons. Surprisingly, selenocysteine incorporation is enhanced. Overexpression of eRF3 did not affect incorporation efficiency. Coexpression of both factors reproduced the effects with eRF1 alone. Finally, we show that the nucleotide context immediately upstream and downstream of the UGA codon significantly affects termination to incorporation ratios and the response to eRF overexpression. Implications for the mechanisms of selenocysteine incorporation and termination are discussed.


Subject(s)
Peptide Chain Termination, Translational/genetics , Peptide Termination Factors/metabolism , Protein Biosynthesis/genetics , Selenocysteine/metabolism , Base Sequence , Blotting, Western , Cell Line , Codon/genetics , Genes, Reporter/genetics , Humans , Iodide Peroxidase/metabolism , Mutation/genetics , Peptide Termination Factors/genetics , Proteins/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Selenium/metabolism , Selenocysteine/genetics , Selenoproteins , Transfection
7.
J Biol Chem ; 276(32): 30183-7, 2001 Aug 10.
Article in English | MEDLINE | ID: mdl-11425850

ABSTRACT

Types 1 and 3 iodothyronine deiodinases are known to be selenocysteine-containing enzymes. Although a putative human type 2 iodothyronine deiodinase (D2) gene (hDio2) encoding a similar selenoprotein has been identified, basal D2 activity is not selenium (Se)-dependent nor has D2 been labeled with (75)Se. A human mesothelioma cell line (MSTO-211H) has recently been shown to have approximately 40-fold higher levels of hDio2 mRNA than mesothelial cells. Mesothelioma cell lysates activate thyroxine (T(4)) to 3,5,3'-triiodothyronine with typical characteristics of D2 such as low K(m) (T(4)), 1.3 nm, resistance to propylthiouracil, and a short half-life ( approximately 30 min). D2 activity is approximately 30-fold higher in Se-supplemented than in Se-depleted medium. An antiserum prepared against a peptide deduced from the Dio2 mRNA sequence precipitates a (75)Se protein of the predicted 31-kDa size from (75)Se-labeled mesothelioma cells. Bromoadenosine 3'5' cyclic monophosphate increases D2 activity and (75)Se-p31 approximately 2.5-fold whereas substrate (T(4)) reduces both D2 activity and (75)Se-p31 approximately 2-3-fold. MG132 or lactacystin (10 microm), inhibitors of the proteasome pathway by which D2 is degraded, increase both D2 activity and (75)Se-p31 3-4-fold and prevent the loss of D2 activity during cycloheximide or substrate (T(4)) exposure. Immunocytochemical studies with affinity-purified anti-hD2 antibody show a Se-dependent increase in immunofluorescence. Thus, human D2 is encoded by hDio2 and is a member of the selenodeiodinase family accounting for its highly catalytic efficiency in T(4) activation.


Subject(s)
Acetylcysteine/analogs & derivatives , Iodide Peroxidase/biosynthesis , Iodide Peroxidase/physiology , Mesothelioma/enzymology , Proteins/chemistry , Acetylcysteine/pharmacology , Animals , Humans , Immunohistochemistry , Iodide Peroxidase/chemistry , Kinetics , Microscopy, Confocal , Microscopy, Fluorescence , Propylthiouracil/pharmacology , Proteins/physiology , RNA, Messenger/metabolism , Selenium/metabolism , Selenoproteins , Transfection , Tumor Cells, Cultured
8.
Am J Physiol Endocrinol Metab ; 281(1): E54-61, 2001 Jul.
Article in English | MEDLINE | ID: mdl-11404222

ABSTRACT

The goal of the present investigation was to analyze the types 2 (D2) and 3 (D3) iodothyronine deiodinases in various structures within the central nervous system (CNS) in response to iodine deficiency. After 5-6 wk of low-iodine diet (LID) or LID + 2 microg potassium iodide/ml (LID + KI; control), rats' brains were processed for in situ hybridization histochemistry for D2 and D3 mRNA or dissected, frozen in liquid nitrogen, and processed for D2 and D3 activities. LID did not affect weight gain or serum triiodothyronine, but plasma thyroxine (T4) was undetectable. In the LID + KI animals, D3 activities were highest in the cerebral cortex (CO) and hippocampus (HI), followed by the olfactory bulb and was lowest in cerebellum (CE). Iodine deficiency decreased D3 mRNA expression in all CNS regions, and these changes were accompanied by three- to eightfold decreases in D3 activity. In control animals, D2 activity in the medial basal hypothalamus (MBH) was similar to that in pituitary gland. Of the CNS D2-expressing regions analyzed, the two most responsive to iodine deficiency were the CO and HI, in which an approximately 20-fold increase in D2 activity occurred. Other regions, i.e., CE, lateral hypothalamus, MBH, and pituitary gland, showed smaller increases. The distribution of and changes in D2 mRNA were similar to those of D2 activity. Our results indicate that decreases in the expression of D3 and increases in D2 are an integral peripheral component of the physiological response of the CNS to iodine deficiency.


Subject(s)
Adaptation, Physiological/physiology , Central Nervous System/physiology , Iodide Peroxidase/metabolism , Iodine/deficiency , Animals , Diet , In Situ Hybridization , Iodide Peroxidase/biosynthesis , Isoenzymes/metabolism , Male , Organ Size/physiology , RNA, Messenger/biosynthesis , Rats , Rats, Sprague-Dawley , Thyroid Gland/pathology , Thyroid Hormones/blood
9.
Mol Cell Biol ; 21(11): 3840-52, 2001 Jun.
Article in English | MEDLINE | ID: mdl-11340175

ABSTRACT

Selenocysteine (Sec) tRNA (tRNA([Ser]Sec)) serves as both the site of Sec biosynthesis and the adapter molecule for donation of this amino acid to protein. The consequences on selenoprotein biosynthesis of overexpressing either the wild type or a mutant tRNA([Ser]Sec) lacking the modified base, isopentenyladenosine, in its anticodon loop were examined by introducing multiple copies of the corresponding tRNA([Ser]Sec) genes into the mouse genome. Overexpression of wild-type tRNA([Ser]Sec) did not affect selenoprotein synthesis. In contrast, the levels of numerous selenoproteins decreased in mice expressing isopentenyladenosine-deficient (i(6)A(-)) tRNA([Ser]Sec) in a protein- and tissue-specific manner. Cytosolic glutathione peroxidase and mitochondrial thioredoxin reductase 3 were the most and least affected selenoproteins, while selenoprotein expression was most and least affected in the liver and testes, respectively. The defect in selenoprotein expression occurred at translation, since selenoprotein mRNA levels were largely unaffected. Analysis of the tRNA([Ser]Sec) population showed that expression of i(6)A(-) tRNA([Ser]Sec) altered the distribution of the two major isoforms, whereby the maturation of tRNA([Ser]Sec) by methylation of the nucleoside in the wobble position was repressed. The data suggest that the levels of i(6)A(-) tRNA([Ser]Sec) and wild-type tRNA([Ser]Sec) are regulated independently and that the amount of wild-type tRNA([Ser]Sec) is determined, at least in part, by a feedback mechanism governed by the level of the tRNA([Ser]Sec) population. This study marks the first example of transgenic mice engineered to contain functional tRNA transgenes and suggests that i(6)A(-) tRNA([Ser]Sec) transgenic mice will be useful in assessing the biological roles of selenoproteins.


Subject(s)
Protein Biosynthesis , Proteins , RNA, Transfer, Amino Acid-Specific/biosynthesis , Animals , Base Sequence , Blotting, Northern/methods , Gene Expression , Isopentenyladenosine/genetics , Isopentenyladenosine/metabolism , Mice , Mice, Transgenic , Molecular Sequence Data , Nucleic Acid Conformation , Selenium/metabolism , Selenoproteins
10.
Mol Endocrinol ; 15(1): 112-24, 2001 Jan.
Article in English | MEDLINE | ID: mdl-11145743

ABSTRACT

Types 1 and 2 iodothyronine deiodinases (D1 and D2) catalyze the production of T(3) from T(4). D2 mRNA is abundant in the human thyroid but very low in adult rat thyroid, whereas D1 activity is high in both. To understand the molecular regulation of these genes in thyroid cells, the effect of thyroid transcription factor 1 (TTF-1) and the paired domain-containing protein 8 (Pax-8) on the transcriptional activity of the deiodinase promoters were studied. Both the approximately 6.5-kb hdio2 sequence and its most 3' 633 bp were activated 10-fold by transiently expressed TTF-1 in COS-7 cells, but the hdio1 was unaffected. Surprisingly, the response of the rdio2 gene to TTF-1 was only 3-fold despite the 73% identity with the proximal 633-bp region of hdio2 including complete conservation of a functional cAMP response element at -90. Neither human nor rat dio2 nor human dio1 was induced by Pax-8. The binding affinity of four putative TTF-1 binding sites in hdio2 were compared by a semiquantitative gel retardation assay using in vitro expressed TTF-1 homeodomain protein. Only two sites, D and C1 (both of which are absent in rdio2), had significant affinity. Functional analyses showed that both sites are required for the full response to TTF-1. These results can explain the differential expression of dio2 in thyroid and potentially other tissues in humans and rats.


Subject(s)
Gene Expression/drug effects , Iodide Peroxidase/genetics , Nuclear Proteins/pharmacology , Thyroid Gland/enzymology , Transcription Factors/pharmacology , Animals , Base Sequence , Binding Sites , Cell Line , Cyclic AMP-Dependent Protein Kinases/pharmacology , DNA/chemistry , DNA/metabolism , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/pharmacology , Gene Library , Humans , Iodide Peroxidase/metabolism , Iodine Radioisotopes , Kinetics , Male , Molecular Sequence Data , Nuclear Proteins/metabolism , PAX8 Transcription Factor , Paired Box Transcription Factors , Promoter Regions, Genetic , Propylthiouracil/pharmacology , RNA, Messenger/analysis , Rats , Rats, Sprague-Dawley , Sequence Homology , Species Specificity , Thyroid Nuclear Factor 1 , Thyroxine/metabolism , Trans-Activators/metabolism , Trans-Activators/pharmacology , Transcription Factors/metabolism , Transcription, Genetic/drug effects
11.
Endocrinology ; 141(12): 4606-12, 2000 Dec.
Article in English | MEDLINE | ID: mdl-11108274

ABSTRACT

Human type 2 iodothyronine deiodinase (hD2) catalyzes the activation of T4 to T3. D2, like types 1 and 3 deiodinases, contains selenocysteine (Sec) in the highly conserved active center at position 133. To evaluate the contribution of Sec133 to the catalytic properties of hD2, we generated mutants in which cysteine (Cys) or alanine (Ala) replaced Sec133. The Km (T4) of Cys133D2 was 2.1 microM, strikingly higher than that of native D2 (1.4 nM). In contrast, the relative turnover number was 10-fold lower for Cys133D2, illustrating the greater potency of Se than S in supporting catalysis. The AlaD2 mutant was inactive. Studies in intact cells transiently expressing the native or Cys133D2 enzyme exhibited saturation kinetics expected from the Km as measured under in vitro conditions, indicating rapid equilibration of extracellular and intracellular T4. Blockade of the NTCP, OATP1-3, and LST-1 transporters with 10 mM sodium taurocholate did not alter the deiodination rate of T4 by Cys133D2 transiently expressed in intact cells, suggesting that intracellular transport of T4 is not rate limiting. These results illustrate that selenium plays a critical role in deiodination catalyzed by hD2.


Subject(s)
Iodide Peroxidase/chemistry , Iodide Peroxidase/metabolism , Selenocysteine , Alanine , Catalysis , Cell Line , Cysteine , Gene Expression , Humans , Iodide Peroxidase/genetics , Iodine Radioisotopes , Kinetics , Mutagenesis , Recombinant Proteins , Structure-Activity Relationship , Thyroxine/metabolism , Transfection , Triiodothyronine/metabolism
12.
Genes Cells ; 5(11): 897-903, 2000 Nov.
Article in English | MEDLINE | ID: mdl-11122377

ABSTRACT

BACKGROUND: Selenoprotein P is a protein of considerable intrigue, due to its unusual composition and requirements for its biosynthesis. Whereas most selenoproteins contain a single selenocysteine residue, the human, bovine and rodent selenoprotein P genes encode proteins containing 10-12 selenocysteines. Selenoprotein P genes have, to date, only been reported in mammals, and the function of the protein remains elusive. RESULTS: Herein, we report the identification and characterization of nonmammalian selenoprotein P in the zebrafish Danio rerio. Sequencing of the cDNA revealed the presence of 17 selenocysteine codons, the highest number reported in any protein. Two histidine-rich regions present in the mammalian selenoprotein P sequences are conserved in the zebrafish protein, and two SECIS elements are present in the 3' untranslated region. Whole-mount in situ hybridization of zebrafish embryos revealed high levels of expression of selenoprotein P mRNA in fertilized eggs and in the yolk sac of developing embryos. Transient transfection of the cDNA in mammalian cells resulted in efficient expression of the full-length secreted selenoprotein. A single N-glycosylation site is predicted, and shown to be utilized. CONCLUSIONS: Discovery of selenoprotein P in the zebrafish opens a previously unavailable avenue for genetic investigation of the functions of this unusual protein.


Subject(s)
Protein Biosynthesis , Proteins/genetics , Amino Acid Sequence/genetics , Animals , Cell Line , Gene Expression , Glycosylation , Humans , In Situ Hybridization , Molecular Sequence Data , Organ Specificity , Physical Chromosome Mapping , Proteins/chemistry , RNA, Messenger/biosynthesis , Selenium Radioisotopes , Selenoprotein P , Selenoproteins , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Transfection , Zebrafish , Zebrafish Proteins
13.
EMBO J ; 19(24): 6882-90, 2000 Dec 15.
Article in English | MEDLINE | ID: mdl-11118223

ABSTRACT

Selenocysteine incorporation at UGA codons requires cis-acting mRNA secondary structures and several specialized trans-acting factors. The latter include a selenocysteine-specific tRNA, an elongation factor specific for this tRNA and a SECIS-binding protein, SBP2, which recruits the elongation factor to the selenoprotein mRNA. Overexpression of selenoprotein mRNAs in transfected cells results in inefficient selenocysteine incorporation due to limitation of one or more of these factors. Using a transfection-based competition assay employing overexpression of selenoprotein mRNAs to compete for selenoprotein synthesis, we investigated the ability of the trans-acting factors to overcome competition and restore selenocysteine incorporation. We report that co-expression of SBP2 overcomes the limitation produced by selenoprotein mRNA overexpression, whereas selenocysteyl-tRNA and the selenocysteine-specific elongation factor do not. Competition studies indicate that once bound to SECIS elements, SBP2 does not readily exchange between them. Finally, we show that SBP2 preferentially stimulates incorporation directed by the seleno protein P and phospholipid hydroperoxide glutathione peroxidase SECIS elements over those of other selenoproteins. The mechanistic implications of these findings for the hierarchy of selenoprotein synthesis and nonsense-mediated decay are discussed.


Subject(s)
Proteins , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/metabolism , Selenocysteine/genetics , Cell Line , Genes, Reporter , Humans , Iodide Peroxidase/genetics , Iodide Peroxidase/metabolism , Mutagenesis, Site-Directed , Open Reading Frames , Protein Biosynthesis , RNA, Messenger/genetics , RNA-Binding Proteins/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Selenocysteine/metabolism , Selenoproteins , Transcription, Genetic , Transfection , beta-Galactosidase/genetics , beta-Galactosidase/metabolism
14.
Mol Endocrinol ; 14(11): 1697-708, 2000 Nov.
Article in English | MEDLINE | ID: mdl-11075806

ABSTRACT

We investigated the mechanism by which T4 regulates its activation to T3 by the type 2 iodothyronine deiodinase (D2). D2 is a short- lived (t1/2 50 min), 31-kDa endoplasmic reticulum (ER) integral membrane selenoenzyme that generates intracellular T3. Inhibition of the ubiquitin (Ub) activating enzyme, E1, or MG132, a proteasome blocker, inhibits both the basal and substrate-induced acceleration of D2 degradation. Using a catalytically active transiently expressed FLAG-tagged-NH2-D2, we found rapid synthesis of high molecular mass (100-300 kDa) Ub-D2 conjugates that are catalytically inactive. Ub-D2 increases when cells are exposed to D2 substrate or MG132 and disappears rapidly after E1 inactivation. Fusion of FLAG epitope to the COOH terminus of D2 prolongs its half-life approximately 2.5-fold and increases the levels of active and, especially, Ub-D2. This indicates that COOH-terminal modification interferes with proteasomal uptake of Ub-D2 that can then be deubiquitinated. Interestingly, the type 1 deiodinase, a related selenoenzyme that also converts T4 to T3 but with a half-life of >12 h, is inactivated but not ubiquitinated or degraded after exposure to substrate. Thus, ubiquitination of the ER-resident enzyme D2 constitutes a specific posttranslational mechanism for T4 regulation of its own activation in the central nervous system and pituitary tissues in which D2-catalyzed T4 to T3 conversion is the major source of intracellular T3.


Subject(s)
Cysteine Endopeptidases/metabolism , Hormones/metabolism , Iodide Peroxidase/metabolism , Multienzyme Complexes/metabolism , Ubiquitins/metabolism , Animals , Base Sequence , Cell Line , Endoplasmic Reticulum/metabolism , Epitopes/genetics , Humans , Iodide Peroxidase/genetics , Ligases/metabolism , Molecular Sequence Data , Oligopeptides , Peptides/genetics , Peptides/immunology , Peptides/metabolism , Proteasome Endopeptidase Complex , Protein Processing, Post-Translational , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Thyroxine/metabolism , Triiodothyronine/metabolism , Ubiquitin-Protein Ligases , Iodothyronine Deiodinase Type II
15.
Endocrinology ; 141(11): 4309-12, 2000 Nov.
Article in English | MEDLINE | ID: mdl-11089566

ABSTRACT

We compared the subcellular localization of FLAG-epitope tagged Types 1 and 2 deiodinases (D1 and D2) transiently expressed in human embryonic kidney (HEK-293) and mouse neuroblastoma (NB2A) cells. D2 is an integral membrane protein based on resistance to extraction at pH 11 with the NH2 terminus in the endoplasmic reticulum (ER). Immunofluorescence confocal microscopy using anti-FLAG and anti-GRP78/BiP antibodies showed the FLAG-D1 signal was found in the periphery of the cells and not co-localized with the ER specific marker GRP78/BiP. On the other hand, FLAG-D2 protein was found in the ER co-localized with the GRP78/BiP protein. These differential distribution patterns indicate subcellular sorting of D1 and D2 is determined by intrinsic protein sequence and can explain the ready access of D2-generated T3 to the nucleus.


Subject(s)
Fluorescent Antibody Technique , Iodide Peroxidase/analysis , Isoenzymes/analysis , Microscopy, Confocal , Subcellular Fractions/enzymology , Animals , Cell Line , Cell Membrane/enzymology , Cell Membrane Permeability/drug effects , Digitonin/pharmacology , Endoplasmic Reticulum/enzymology , Endoplasmic Reticulum Chaperone BiP , Gene Expression , Humans , Iodide Peroxidase/genetics , Isoenzymes/genetics , Kidney/enzymology , Kidney/ultrastructure , Mice , Neuroblastoma/enzymology , Neuroblastoma/ultrastructure , Transfection , Tumor Cells, Cultured
17.
J Biol Chem ; 275(9): 6288-94, 2000 Mar 03.
Article in English | MEDLINE | ID: mdl-10692426

ABSTRACT

Most selenoproteins contain a single selenocysteine residue per polypeptide chain, encoded by an in-frame UGA codon. Selenoprotein P is unique in that its mRNA encodes 10-12 selenocysteine residues, depending on species. In addition to the high number of selenocysteines, the protein is cysteine- and histidine-rich. The function of selenoprotein P has remained elusive, in part due to the inability to express the recombinant protein. This has been attributed to presumed inefficient translation through the selenocysteine/stop codons. Herein, we report for the first time the expression of recombinant rat selenoprotein P in a transiently transfected human epithelial kidney cell line, as well as the endogenously expressed protein from HepG2 and Chinese hamster ovary cells. The majority of the expressed protein migrates with the predicted 57-kDa size of full-length glycosylated selenoprotein P. Based on the histidine-rich nature of selenoprotein P, we have purified the recombinant and endogenously expressed proteins using nickel-agarose affinity chromatography. We show that the recombinant rat and endogenous human proteins react in Western blotting and immunoprecipitation assays with commercial anti-histidine antibodies. The ability to express, purify, and immunochemically detect the recombinant protein provides a foundation for investigating the functions and efficiency of expression of this intriguing protein.


Subject(s)
Proteins/genetics , Selenocysteine/metabolism , Animals , Antibodies/metabolism , Blotting, Western , Cell Line , Chromatography, Affinity , Gene Expression , Glycosylation , Histidine/immunology , Humans , Precipitin Tests , Proteins/immunology , Proteins/isolation & purification , RNA, Messenger/metabolism , Recombinant Proteins , Selenium Radioisotopes , Selenoprotein P , Selenoproteins
18.
EMBO Rep ; 1(2): 158-63, 2000 Aug.
Article in English | MEDLINE | ID: mdl-11265756

ABSTRACT

Decoding UGA as selenocysteine requires a unique tRNA, a specialized elongation factor, and specific secondary structures in the mRNA, termed SECIS elements. Eukaryotic SECIS elements are found in the 3' untranslated region of selenoprotein mRNAs while those in prokaryotes occur immediately downstream of UGA. Consequently, a single eukaryotic SECIS element can serve multiple UGA codons, whereas prokaryotic SECIS elements only function for the adjacent UGA, suggesting distinct mechanisms for recoding in the two kingdoms. We have identified and characterized the first eukaryotic selenocysteyl-tRNA-specific elongation factor. This factor forms a complex with mammalian SECIS binding protein 2, and these two components function together in selenocysteine incorporation in mammalian cells. Expression of the two functional domains of the bacterial elongation factor-SECIS binding protein as two separate proteins in eukaryotes suggests a mechanism for rapid exchange of charged for uncharged selenocysteyl-tRNA-elongation factor complex, allowing a single SECIS element to serve multiple UGA codons.


Subject(s)
3' Untranslated Regions/genetics , Nucleic Acid Conformation , Peptide Elongation Factors/metabolism , Proteins , RNA, Transfer, Amino Acyl/genetics , Regulatory Sequences, Nucleic Acid , Selenocysteine/genetics , Amino Acid Sequence , Animals , Cell Line , Humans , Mice , Molecular Sequence Data , Peptide Elongation Factors/chemistry , Peptide Elongation Factors/genetics , Protein Biosynthesis , RNA, Transfer, Amino Acyl/metabolism , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Rats , Selenocysteine/metabolism , Selenoproteins , Sequence Alignment , Transfection
19.
Endocrinology ; 141(1): 229-37, 2000 Jan.
Article in English | MEDLINE | ID: mdl-10614643

ABSTRACT

The type 2 iodothyronine deiodinase (D2) catalyzes T4 activation. In humans, unlike rodents, it is widely expressed, and its action probably contributes to both intracellular and plasma T3 pools. We have isolated the 6.5-kb 5'-flanking region (FR) and the previously uncloned 553 nucleotides (nt) of the 5'-untranslated region (UTR) of hdio2. The 5'-UTR is complex, with three transcription start sites (TSS) (708, 31, and approximately 24 nt 5' to the ATG), an alternatively spliced approximately 300-nt intron in the 5'-UTR, and three short open reading frames 5' to the initiator ATG. The previously reported approximately 7.5-kb D2 messenger RNA (mRNA) is actually an approximately 7-kb doublet that is present in thyroid, pituitary, cardiac and skeletal muscle, and possibly brain, but with only the longer transcript in placenta. A canonical cAMP response element-binding protein-binding site is present at about 90 bp 5' to the most 5'-TSS. It accounts for the robust response of the 6.8-kb hdio2 5'-FR to protein kinase A. Forskolin increases D2 mRNA in human thyroid cells, which may explain the high D2 mRNA in Graves' thyroid and thyroid adenomas. The hdio2 gene structure and Northern blot results suggest that D2 expression is tightly controlled and tissue specific.


Subject(s)
5' Untranslated Regions/genetics , Cyclic AMP/physiology , Endopeptidases , Iodide Peroxidase/genetics , Adenoma/metabolism , Adenoma/pathology , Base Sequence , Chloramphenicol O-Acetyltransferase/biosynthesis , Chloramphenicol O-Acetyltransferase/genetics , Codon, Initiator/genetics , Colforsin/isolation & purification , Colforsin/pharmacology , DNA Primers/genetics , Exopeptidases/metabolism , Humans , Introns/genetics , Molecular Sequence Data , Promoter Regions, Genetic/genetics , RNA, Messenger/biosynthesis , TATA Box/genetics , Thyroid Neoplasms/metabolism , Thyroid Neoplasms/pathology , Thyrotropin/pharmacology , Transcription, Genetic , Tumor Cells, Cultured , Iodothyronine Deiodinase Type II
20.
J Biol Chem ; 274(36): 25379-85, 1999 Sep 03.
Article in English | MEDLINE | ID: mdl-10464265

ABSTRACT

Thioredoxin reductases function in regulating cellular redox and function through their substrate, thioredoxin, in the proper folding of enzymes and redox regulation of transcription factor activity. These enzymes are overexpressed in certain tumors and cancer cells and down-regulated in apoptosis and may play a role in regulating cell growth. Mammalian thioredoxin reductases contain a selenocysteine residue, encoded by a UGA codon, as the penultimate carboxyl-terminal amino acid. This amino acid has been proposed to carry reducing equivalents from the active site to substrates. We report expression of a wild-type thioredoxin reductase selenoenzyme, a cysteine mutant enzyme, and the UGA-terminated protein in mammalian cells and overexpression of the cysteine mutant and UGA-terminated proteins in the baculovirus insect cell system. We show that substitution of cysteine for selenocysteine decreases enzyme activity for thioredoxin by 2 orders magnitude, and that termination at the UGA codon abolishes activity. We further demonstrate the presence of a functional selenocysteine insertion sequence element that is highly active but only moderately responsive to selenium supplementation. Finally, we show that thioredoxin reductase mRNA levels are down-regulated by other sequences in the 3'-untranslated region, which contains multiple AU-rich instability elements. These sequences are found in a number of cytokine and proto-oncogene mRNAs and have been shown to confer rapid mRNA turnover.


Subject(s)
Gene Expression Regulation, Enzymologic , RNA, Messenger/genetics , Thioredoxin-Disulfide Reductase/genetics , 3' Untranslated Regions , Cell Line , DNA Transposable Elements , Humans , Proto-Oncogene Mas , Selenocysteine/genetics , Thioredoxin-Disulfide Reductase/biosynthesis
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