Species differences in liver type I iodothyronine deiodinase.

The type I iodothyronine deiodinase (ID-I) of liver is an important enzyme for the conversion of the prohormone thyroxine (T4) to the active thyroid hormone 3,3',5-triiodothyronine (T3). Because it is an integral membrane protein of low abundance, purification of ID-I from rat liver has proven to be difficult. We have analyzed ID-I in liver microsomal fractions from various animals to reveal possible species differences and to explore alternative sources for the isolation of the enzyme. ID-I was characterized by enzyme assay with 3,3',5'-triiodothyronine (rT3) as the preferred substrate and by affinity-labeling with N-bromoacetyl-[125I]T3 (BrAc[125I]T3). Labeled ID-I subunit was identified and quantified by SDS-PAGE and autoradiography. The Mr of ID-I in the species investigated varied between 25.7 and 29.1 kDa. Rat and dog liver microsomes had a markedly higher enzyme content than microsomes of human, mouse, rabbit, cow, pig, sheep, goat, chicken or duck liver. Rat liver microsomes showed the highest ID-I activity of all species examined. Turnover numbers for ID-I varied between 264 and 1059 min-1, with rabbit and goat showing the highest values. However, dog liver ID-I displayed an exceptionally low turnover number of 78 min-1. In conclusion, ID-I has similar properties in all species examined with the notable exception of dog.     

Selenouracil derivatives are potent inhibitors of the selenoenzyme type I iodothyronine deiodinase.

Type I iodothyronine deiodinase (ID-I) is a selenoenzyme, which is important for the conversion of the prohormone thyroxine (T4) to the bioactive thyroid hormone 3,3',5-triiodothyronine (T3). 2-Thiouracil derivatives inhibit ID-I by interaction with an enzyme form generated during catalysis. We have now tested the potential inhibitory effects of the selenocompounds 6-methyl- (MSU) and 6-propyl-2-selenouracil (PSU) in comparison with their thioanalogs 6-methyl- (MTU) and 6-propyl-2-thiouracil (PTU) on rat liver ID-I activity using 3,3',5-triiodothyronine (reverse T3, rT3) as substrate and dithiothreitol (DTT) as cofactor. All compounds showed dose-dependent inhibition of ID-I with IC50 values of 1, 0.5, 0.4 and 0.2 microM for MTU, MSU, PTU and PSU, respectively. Our results further suggest that these inhibitions are uncompetitive with substrate and competitive with cofactor. The high potency of selenouracils may be due to reaction with a substrate-induced enzyme selenenyl iodide intermediate under formation of a stable enzyme-selenouracil diselenide.     

Identification of essential histidine residues in rat type I iodothyronine deiodinase.

Deiodination is required for conversion of thyroxine, the inactive prohormone secreted by the thyroid gland, to 3,5,3'-triiodothyronine, the biologically active thyroid hormone. The principal enzyme catalyzing this reaction, Type I iodothyronine 5' deiodinase, was shown recently to contain the amino acid, selenocysteine, and site-directed mutagenesis showed that this amino acid confers the biochemical properties characteristic of this enzyme. Previous studies suggest that a histidine residue may also be critical for activity. To further our understanding of the biochemical mechanism of this reaction, we have used in vitro mutagenesis to examine the contribution of each of the 4 histidines in this enzyme to the deiodination process. Two of the histidines (185 and 253) are not involved in deiodination, as their removal had no effect on activity. Mutagenesis of histidine 158 resulted in complete loss of activity, suggesting a role in either protein conformation or catalysis. The most informative results were obtained from the studies of histidine 174. Mutagenesis of this histidine to asparagine or glutamine altered reactivity with substrate and reduced inhibition by diethylpyrocarbonate and rose bengal. These results demonstrate that histidine 174 is critical to function and appears to be involved in binding of hormone.     

Characterization of the subunit structure of the catalytically active type I iodothyronine deiodinase

Type I iodothyronine deiodinase is a approximately 50-kDa, integral membrane protein that catalyzes the outer ring deiodination of thyroxine. Despite the identification and cloning of a 27-kDa selenoprotein with the catalytic properties of the type I enzyme, the composition and the physical nature of the active deiodinase are unknown. In this report, we use a molecular approach to determine holoenzyme composition, the role of the membrane anchor on enzyme assembly, and the contribution of individual 27-kDa subunits to catalysis. Overexpression of an immunologically unique rat 27-kDa protein in LLC-PK1 cells that contain abundant catalytically active 27-kDa selenoprotein decreased deiodination by approximately 50%, and > 95% of the LLC-PK1 derived 27-kDa selenoprotein was specifically immune precipitated by the anti-rat enzyme antibody. The hybrid enzyme had a molecular mass of 54 kDa and an s(20,w) of approximately 3.5 S indicating that every native 27-kDa selenoprotein partnered with an inert rat 27-kDa subunit in a homodimer. Enzyme assembly did not depend on the presence of the N-terminal membrane anchor of the 27-kDa subunit. Direct visualization of the deiodinase dimer showed that the holoenzyme was sorted to the basolateral plasma membrane of the renal epithelial cell.     

Rat liver type I iodothyronine deiodinase is not identical to protein disulfide isomerase

This study was done to test the recent hypothesis (Boado et al. (1988) Biochem. Biophys. Res. Commun. 155, 1297-1304) that type I iodothyronine deiodinase (ID-I) is identical to protein disulfide isomerase (PDI). Autoradiograms of rat liver microsomal proteins, labeled with N-bromoacetyl-[125I]triiodothyronine (BrAc[125I]T3) and separated by SDS-PAGE, show predominantly 2 radioactive bands of Mr 27 and 56 kDa. Substrates and inhibitors of ID-I inhibited labeling of the 27 kDa band but not that of the 56 kDa band. Treatment of microsomes with trypsin abolished labeling of the 27 kDa protein and destroyed the activity of ID-I but did not prevent labeling of the 56 kDa protein. Following treatment of microsomes at pH 8.0-9.5 or with 0.05% deoxycholate (DOC) PDI content and labeling of the 56 kDa protein were strongly diminished but ID-I activity and labeling of the 27 kDa protein were not affected. The latter decreased in parallel after treatment at pH greater than or equal to 10. Rat pancreas microsomes contain high amounts of PDI but show no ID-I activity. Reaction of these microsomes with BrAc[125I]T3 results in extensive labeling of a 56 kDa protein but no labeling of a 27 kDa protein. Pure PDI (Mr 56 kDa) was readily labeled by BrAc[125I]T3 but showed no deiodinase activity. These results strongly suggest that the 27 kDa band represents (a subunit of) ID-I while the 56 kDa band represents PDI. From these and other data it is concluded that PDI and ID-I are not identical proteins.     

Inhibition of rat liver iodothyronine deiodinase. Interaction of aurones with the iodothyronine ligand-binding site

We report that aurone derivatives of plant extracts produce potent, dose-dependent, and ultimately complete inhibition of three different metabolic monodeiodination pathways catalyzed by rat liver microsomal type I iodothyronine deiodinase. These data show that (3'),4',4,6-(tetra)trihydroxyaurones are the most potent naturally occurring plant-derived inhibitors of this deiodinase enzyme (IC50 V 0.5 microM). Lineweaver-Burk analysis using both L-thyroxine (T4) and 3',5',3-triiodothyronine as substrates suggests a cofactor competitive mechanism of inhibition for 4',4,6-trihydroxyaurone which also can displace 125I-L-T4 from binding to thyroxine-binding prealbumin with a potency comparable to its inhibition of T4-5'-deiodinase. Among type I deiodinase inhibitors, cofactor competition has been observed only for propylthiourea. Computer graphic modeling studies were also carried out to explore aurone conformations and to compare them with those of the thyroid hormones. This analysis shows that the aurones can adopt either a planar or an antiskewed conformation, such as observed for 3',5',3-triiodothyronine, the most potent natural deiodinase substrate inhibitor. The thyroxine-binding prealbumin complex was used to model the deiodinase ligand binding site because of the similarity observed between inhibitor binding affinity and enzyme inhibition characteristics. These studies show that the aurones which adopt an antiskewed conformation can interact favorably in the prealbumin binding site. This model of the deiodinase active site can be used to design other deiodinase inhibitors.     

Inhibition of type I and type II iodothyronine deiodinase activity in rat liver, kidney and brain produced by selenium deficiency.

Selenium deficiency for periods of 5 or 6 weeks in rats produced an inhibition of tri-iodothyronine (T3) production from added thyroxine (T4) in brain, liver and kidney homogenate. This inhibition was reflected in plasma T4 and T3 concentrations, which were respectively increased and decreased in selenium-deficient animals. Although plasma T4 levels increased in selenium-deficient animals, this did not produce the normal feedback inhibition on thyrotropin release from the pituitary. Selenium deficiency was confirmed in the animals by decreased selenium-dependent glutathione peroxidase (Se-GSH-Px) activity in all of these tissues. Administration of selenium, as a single intraperitoneal injection of 200 micrograms of selenium (as Na2SeO3)/kg body weight completely reversed the effects of selenium deficiency on thyroid-hormone metabolism and partly restored the activity of Se-GSH-Px. Selenium administration at 10 micrograms/kg body weight had no significant effect on thyroid-hormone metabolism or on Se-GSH-Px activity in any of the tissues studied. The characteristic changes in plasma thyroid-hormone levels that occurred in selenium deficiency appeared not to be due to non-specific stress factors, since food restriction to 75% of normal intake or vitamin E deficiency produced no significant changes in plasma T4 or T3 concentration. These data are consistent with the view that the Type I and Type II iodothyronine deiodinase enzymes are seleno-enzymes or require selenium-containing cofactors for activity.     

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

The 5'-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'-iodothyronine deiodinase was 55,400 and for the type II enzyme was 198,700. These data indicate that the two isozymes of iodothyronine 5'-deiodinase are multimeric, differ in holoenzyme size and subunit composition, and that their substrate binding subunits are distinct.     

The human type 2 iodothyronine deiodinase is a selenoprotein highly expressed in a mesothelioma cell line.

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.     

Preparation and properties of a selenium-containing catalytic antibody as type I deiodinase mimic

Conversion of thyroxine (T4) to 3,5,3'-triiodothyronine is an essential first step in controlling thyroid hormone action. Type I deiodinase (DI) can catalyze the conversion to produce the bulk of serum 3,5,3'-triiodothyronine. Acting as a mimic of DI, a selenium-containing catalytic antibody (Se-4C5) prepared by converting the serine residues of monoclonal antibody 4C5 raised against T4 into selenocysteines, can catalyze the deiodination of T4 with dithiothreitol (DTT) as cosubstrate. The mimic enzyme Se-4C5 exhibited a much greater deiodinase activity than model compound ebselen and another selenium-containing antibody Se-Hp4 against GSH. The coupling of selenocysteine with the combining pocket of antibody 4C5 endowed Se-4C5 with enzymatic activity. To probe the catalytic mechanism of the catalytic antibody, detailed kinetic studies were carried out in this paper. Investigations into the deiodinative reaction revealed the relationship between the initial velocity and substrate concentration. The characteristic parallel Dalziel plots demonstrated that Se-4C5-catalyzed reaction mechanism was ping-pong one, involving at least one covalent enzyme intermediate. The kinetic properties of the catalytic antibody were similar to those of DI, with Km values for T4 and DTT of approximately 0.8 microm and 1.8 mm, respectively, and a Vm value of 270 pmol per mg of protein per min. The activity could be sensitively inhibited by 6-propyl-2-thiouracil (PTU) with a K(i) value of approximately 120 microm at 2.0 microm T4 concentration. The PTU inhibition was progressively alleviated with the increasing concentration of added DTT, revealing that PTU was a competitive inhibitor for DTT.     

Sex steroids modulate rat anterior pituitary and liver iodothyronine deiodinase activities.

In this study, we investigated the sex hormone regulation of 5'-iodothyronine deiodinase activity, which is responsible for enzymatic conversion of thyroxine into the bioactive form, triiodothyronine. Pituitary homogenates and liver microsomes from: 1) ovariectomized rats injected with 17-beta-estradiol benzoate and/or progesterone (0.7 and 250 microg/100 g body weight, respectively, subcutaneously, over 10 days); 2) male castrated rats treated or not with 0.4 mg/100 g body weight testosterone propionate, intramuscular, over 7 days, were assayed for type 1 and type 2 deiodinase activity in the pituitary. Enzyme activities were measured by release of (125)I from deiodination of (125)I reverse triiodothyronine under varying assay conditions. Estrogen stimulated anterior pituitary and liver type 1 deiodinase activity in ovariectomized rats (45 and 30 %, p < 0.05). Progesterone inhibited the liver enzyme (40 %, p < 0.05), and had no effect on the pituitary, but in both tissues, blocked estrogen stimulatory effect on type 1 deiodinase. In males, testosterone normalized the reduced liver type 1 deiodinase of castrated rats. However, in the pituitary, castration increased (50 %) type 1 deiodinase independent of testosterone treatment, suggesting the existence of a inhibitory testicular regulator of pituitary type 1 enzyme. Treatments did not alter pituitary type 2 deiodinase activity. In conclusion, gonads and sex steroids differentially modulate type 1 deiodinase activity in rat pituitary and liver.     

Role of the type 2 iodothyronine deiodinase (D2) in the control of thyroid hormone signaling

Background

Thyroid hormone signaling is critical for development, growth and metabolic control in vertebrates. Although serum concentration of thyroid hormone is remarkable stable, deiodinases modulate thyroid hormone signaling on a time- and cell-specific fashion by controlling the activation and inactivation of thyroid hormone.

Scope of the review

This review covers the recent advances in D2 biology, a member of the iodothyronine deiodinase family, thioredoxin fold‐containing selenoenzymes that modify thyroid hormone signaling in a time- and cell-specific manner.

Major conclusions

D2-catalyzed T3 production increases thyroid hormone signaling whereas blocking D2 activity or disruption of the Dio2 gene leads to a state of localized hypothyroidism. D2 expression is regulated by different developmental, metabolic or environmental cues such as the hedgehog pathway, the adrenergic- and the TGR5-activated cAMP pathway, by xenobiotic molecules such as flavonols and by stress in the endoplasmic reticulum, which specifically reduces de novo synthesis of D2 via an eIF2a-mediated mechanism. Thus, D2 plays a central role in important physiological processes such as determining T3 content in developing tissues and in the adult brain, and promoting adaptive thermogenesis in brown adipose tissue. Notably, D2 is critical in the T4-mediated negative feed-back at the pituitary and hypothalamic levels, whereby T4 inhibits TSH and TRH expression, respectively. Notably, ubiquitination is a major step in the control of D2 activity, whereby T4 binding to and/or T4 catalysis triggers D2 inactivation by ubiquitination that is mediated by the E3 ubiquitin ligases WSB-1 and/or TEB4. Ubiquitinated D2 can be either targeted to proteasomal degradation or reactivated by deubiquitination, a process that is mediated by the deubiquitinases USP20/33 and is important in adaptive thermogenesis.

General significance

Here we review the recent advances in the understanding of D2 biology focusing on the mechanisms that regulate its expression and their biological significance in metabolically relevant tissues. This article is part of a Special Issue entitled Thyroid hormone signalling.     

Identification of type I iodothyronine 5'-deiodinase as a selenoenzyme

A 27.8 kDa membrane selenoprotein was previously identified in rat thyroid, liver and kidney, the tissues with the highest activities of type I iodothyronine 5'-deiodinase. This membrane enzyme catalyzes the deiodination of L-thyroxine to the biologically active thyroid hormone 3,3',5-triiodothyronine. A decrease in the activity of this enzyme, observed here in the liver of selenium-deficient rats, was found to be due to the absence of a selenium-dependent membrane-bound component. By chemical and enzymatic fragmentation of the 75Se-labeled selenoprotein and of the 27 kDa substrate binding type I 5'-deiodinase subunit, affinity-labeled with N-bromoacetyl-[125I]L-thyroxine, and comparison of the tracer distribution in the peptide fragments the identity of the two proteins was shown. The data indicate that the deiodinase subunit contains one selenium atom per molecule and suggest that a highly reactive selenocysteine is the residue essential for the catalysis of 5'-deiodination. From the results it can be concluded that type I iodothyronine 5'-deiodinase is a selenoenzyme.     

Expression of type II iodothyronine deiodinase marks the time that a tissue responds to thyroid hormone-induced metamorphosis in Xenopus laevis

The thyroid gland synthesizes thyroxine (T4), which passes through the larval tadpole's circulatory system. The enzyme type II iodothyronine deiodinase (D2) converts thyroxine (T4) to the active hormone 3,5,3'-triiodothyronine (T3) in peripheral tissues. An early response to thyroid hormone (TH) in the Xenopus laevis tadpole is the stimulation of cell division in cells that line the brain ventricles, the lumen of the spinal cord, and the limb buds. These cells express constitutively high levels of D2 mRNA. Exogenous T4 induces early DNA synthesis in brain, spinal cord, and limb buds as efficiently as T3. The deiodinase inhibitor iopanoic acid blocks T4- but not T3-induced cell division. At metamorphic climax, both TH-induced cell division and D2 expression decrease in the brain. Then D2 expression appears in late-responding tissues including the anterior pituitary, the intestine, and the tail where cell division is reduced or absent. Therefore, constitutive expression of D2 occurs in the earliest target tissues of TH that will grow and differentiate, while TH-induced expression of D2 takes place in late-responding tissues that will remodel or die. This pattern of constitutive and induced D2 expression contributes to the timing of metamorphic changes in these tissues.