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.     

Metabolism of the thyroid hormones

This review covers the current knowledge about the various metabolic pathways involved in the conversion of thyroid hormones to the thyromimetically active and inactive iodothyronines. The concerted mechanism of systemic and local production of iodothyronines by tissue-specific iodothyronine deiodinase isozymes will ultimately determine the expression of thyroid hormone action. This is exemplified for the regulation of synthesis and release of TSH by iodothyronines at the pituitary level. Iodothyronine metabolites, e.g. Triac, rT3 and T3 amine may modulate TSH secretion, and alterations of local pituitary deiodination (e.g. iopanoate inhibition) influence diurnal TSH secretion without changing TRH-dependent episodic TSH secretion pattern. A summary of structure-activity relationships of greater than 200 naturally occurring and synthetic ligands of rat liver type I iodothyronine deiodinase isozyme propylthiouracil-sensitive) in vitro allows the design of iodothyronine analogues which either serve as specific substrates or antagonists of iodothyronine binding and metabolizing proteins. Furthermore, a complete picture of the ligand-complementary active site of the type I isozyme can be derived. A synthetic 'structurally optimized' iodothyronine-analogue flavonoid inhibitor of the type I deiodinase is able to displace T4 from binding to thyroxine-binding prealbumin and leads to unexpected organ-specific alterations of thyroid hormone metabolism and expression of thyroid hormone actions in an animal model. Therefore, for a complete understanding of thyroid hormone metabolism and action, thyroid hormone transport, cellular compartmentalization, and alternate pathways also have to be considered.     

Kinetics and thiol requirements of iodothyronine 5'-deiodination are tissue-specific in common carp (Cyprinus carpio L.)

Iodothyronine deiodinases determine the biological activity of thyroid hormones. Despite the homology of the catalytic sites of mammalian and teleostean deiodinases, in-vitro requirements for the putative thiol co-substrate dithiothreitol (DTT) vary considerably between vertebrate species. To further our insights in the interactions between the deiodinase protein and its substrates: thyroid hormone and DTT, we measured enzymatic iodothyronine 5′-deiodination, Dio1 and Dio2 mRNA expression, and Dio1 affinity probe binding in liver and kidney preparations from a freshwater teleost, the common carp (Cyprinus carpio L.). Deiodination rates, using reverse T3 (rT3, 3,3′,5′-triiodothyronine) as the substrate, were analysed as a function of the iodothyronine and DTT concentrations. In kidney rT3 5′-deiodinase activity measured at rT3 concentrations up to 10 μM and in the absence of DTT does not saturate appreciably. In the presence of 1 mM DTT, renal rT3 deiodination rates are 20-fold lower. In contrast, rT3 5′-deiodination in liver is potently stimulated by 1 mM DTT. The marked biochemical differences between 5′-deiodination in liver and kidney are not associated with the expression of either Dio1 or Dio2 mRNA since both organs express both deiodinase types. In liver and kidney, DTT stimulates the incorporation of N-bromoacetylated affinity labels in proteins with estimated molecular masses of 57 and 55, and 31 and 28 kDa, respectively. Although primary structures are highly homologous, the biochemistry of carp deiodinases differs markedly from their mammalian counterparts.     

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.     

Characterization of rat iodothyronine sulfotransferases

Sulfation appears to be an important pathway for the reversible inactivation of thyroid hormone during fetal development. The rat is an often used animal model to study the regulation of fetal thyroid hormone status. The present study was done to determine which sulfotransferases (SULTs) are important for iodothyronine sulfation in the rat, using radioactive T4, T3, rT3, and 3,3'-T2 as substrates, 3'-phosphoadenosine-5'-phosphosulfate (PAPS) as cofactor, and rat liver, kidney and brain cytosol, and recombinant rat SULT1A1, -1B1, -1C1, -1E1, -2A1, -2A2, and -2A3 as enzymes. Recombinant rat SULT1A1, -1E1, -2A1, -2A2, and -2A3 failed to catalyze iodothyronine sulfation. For all tissue SULTs and for rSULT1B1 and rSULT1C1, 3,3'-T2 was by far the preferred substrate. Apparent Km values for 3,3'-T2 amounted to 1.9 microM in male liver, 4.4 microM in female liver, 0.76 microM in male kidney, 0.23 microM in male brain, 7.7 microM for SULT1B1, and 0.62 microM for SULT1C1, whereas apparent Km values for PAPS showed less variation (2.0-6.9 microM). Sulfation of 3,3'-T2 was inhibited dose dependently by other iodothyronines, with similar structure-activity relationships for most enzymes except for the SULT activity in rat brain. The apparent Km values of 3,3'-T2 in liver cytosol were between those determined for SULT1B1 and -1C1, supporting the importance of these enzymes for the sulfation of iodothyronines in rat liver, with a greater contribution of SULT1C1 in male than in female rat liver. The results further suggest that rSULT1C1 also contributes to iodothyronine sulfation in rat kidney, whereas other, yet-unidentified forms appear more important for the sulfation of thyroid hormone in rat brain.     

Evidence for a single enzyme in rat liver catalysing the deiodination of the tyrosyl and the phenolic ring of iodothyronines.

The enzymic 5'-deiodination of 3',5'-di-iodothyronine and 5-deiodination of 3,3',5-tri-iodothyronine by rat liver microsomal fractions were found to be characterized by apparent Km values of 0.77 and 17.4 microM respectively, 3',5'-Di-iodothyronine was a competitive inhibitor of 3,3',5-tri-iodothyronine 5-deiodination (Ki 0.65 microM) and 3,3',5-tri-iodothyronine was a competitive inhibitor of 3',5'-di-iodothyronine 5'-deiodination (Ki 19.6 microM). In addition, several radiographic contrast agents and iodothyronine analogues inhibited both reactions competitively and with equal potencies (r = 0.999). These results strongly suggest the existence of a single hepatic deiodinase acting on both the tyrosyl and phenolic ring of iodothyronines.     

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.     

Selenocysteine confers the biochemical properties characteristic of the type I iodothyronine deiodinase.

The conversion of thyroxine to 3,5,3'-triiodothyronine (T3) is the first step in thyroid hormone action, and the Type I iodothyronine deiodinase supplies most of this extrathyroidal T3 in the rat. We found that the cDNA coding for this enzyme contains an in-frame UGA encoding the rare amino acid selenocysteine. Using site-directed mutagenesis, we have converted selenocysteine to cysteine and expressed the wild-type and cysteine mutant enzymes in JEG-3 cells by transient transfection. The kinetic properties of the transiently expressed wild-type enzyme are nearly identical to those reported for rat liver Type I deiodinase. Substitution of sulfur for selenium causes a 10-fold increase in the Km of the enzyme for the favored substrate 3,3',5'-triiodothyronine (rT3), a 100-fold decrease in the sensitivity of rT3 deiodination to competitive inhibition by gold and a 300-fold increase in the apparent Ki for uncompetitive inhibition by 6-n-propylthiouracil. These results demonstrate that selenium is responsible for the biochemical properties which characterize Type I iodothyronine monodeiodination.     

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.     

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.     

Iodothyronine 5-deiodinase in rat posterior pituitary.

We first describe the presence of iodothyronine 5-deiodinase (5D) in the neural lobe of rat pituitary. 6-n-Propyl-2-thiouracil (PTU), a specific inhibitor of type-I deiodinase, had no effect, showing that 5D in neurohypophysis is of type-III isozyme, which is specific for 5-deiodination and has been found only in the brain, placenta and skin. The presence of 5D (type-III) together with our previous report of 5'-deiodinase (type-I in euthyroidism and type-II in hypothyroidism) shows that the isozymes of deiodinases in the neurohypophysis are quite similar to those in the brain. These data suggest a previously unrecognized role of thyroid hormone in posterior pituitary physiology.     

Production and purification of monoclonal T lymphocyte antigen binding molecules (TABM)

The thyroid hormone derivative N-bromoacetyl-3,3',5-triiodothyronine (BrAcT3) acts as an active site-directed inhibitor of rat liver iodothyronine deiodinase. Lineweaver Burk analysis of enzyme kinetic measurements showed that BrAcT3 is a competitive inhibitor of the 5'-deiodination of 3,3',5'-triiodothyronine (rT3) with an apparent Ki value of 0.1 nM. Preincubations of enzyme with BrAcT3 indicated that inhibition by this compound is irreversible. The inactivation rate obeyed saturation kinetics with a limiting inactivation rate constant of 0.35 min-1. Substrates and substrate analogs protected against inactivation by BrAcT3. Covalent incorporation of 125I-labeled BrAcT3 into "substrate-protectable" sites was proportional to the loss of deiodinase activity. The results suggest that BrAcT3 is a very useful affinity label for rat liver iodothyronine deiodinase.     

Plasma thyroxine-binding proteins and thyroid hormone levels in primate species; is callithricidae thyroid hormone resistant?

Thyroxine(T4)-binding to serum proteins in primates; catarrhini, prosimiae, and platyrrhini were studied by polyacrylamide gel electrophoresis T4 binding analysis. From the electrophoretic analysis, it was shown that thyroxine-binding proteins similar to human thyroxine-binding globulin (TBG) and thyroxine-binding prealbumin (TBPA) were present in catarrhini and prosimiae species, but not in platyrrhini (callithricidae and cebidae). T4-binding analysis also revealed that catarrhini and prosimiae have a high affinity T4-binding protein similar to human TBG. The association constant (Ka) for T4 of the plasma proteins in these species was approximately 2.0 X 10(10) M-1. On the other hand, it was unable to demonstrate a high affinity binding site for T4 in the plasma of platyrrhini species. Both the total and free thyroid hormone concentrations in catarrhini and prosimiae were similar to those in human. Total T4 in cebidae, one of the platyrrhini species, was extremely low. Among 8 animals examined, T4 in 6 was undetectable by radioimmunoassay and the mean T4 of the other two was 2.8 micrograms/dl. However, free thyroid hormone concentrations were similar to those in human. In callithricidae, another platyrrhini species, T4 in plasma was 6.90 +/- 2.11, which is comparable to the level in normal human subjects. However, in this species, high-affinity T4-binding protein was lacking and free thyroid hormone concentrations were extremely high (most were higher than the assay limit). Although the thyroid function of callithricidae remains to be studied, it will be interesting if callithricidae is resistant to thyroid hormone action.     

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.     

Identification of a 27-kDa protein with the properties of type II iodothyronine 5'-deiodinase in dibutyryl cyclic AMP-stimulated glial cells

Type II iodothyronine 5'-deiodinase (5'D-II) catalyzes the intracellular conversion of thyroxine (T4) to 3,5,3'-triiodothyronine (T3), producing greater than 90% of the bioactive thyroid hormone in the cerebral cortex. In cultured glial cells, expression of this enzyme is cAMP dependent. Exploiting the cAMP-dependent nature of this enzyme in these cells and utilizing N-bromoacetyl-L-3'- or 5'-[125I]thyroxine (BrAc[125I]T4) to affinity label cellular proteins, a 27-kDa protein with the properties of this enzyme was identified. Intact cells labeled with BrAc[125I]T4 showed three prominent radiolabeled bands of proteins of Mr 55,000, 27,000, and 18,000 (p55, p27, p18, respectively) which incorporated approximately 80% of the affinity label. All three affinity-labeled proteins were membrane associated. One protein (p27) increased 5-6-fold after treating the cells for 16 h with dibutyryl cAMP; maximal specific incorporation of affinity label into the stimulated p27 was approximately 2 pmol/mg of cell protein in intact cells. Alterations in the steady-state levels of 5'D-II resulted in parallel changes in the quantity of p27. In cell sonicates, the rate of enzyme inactivation by BrAcT4 equaled the rate of affinity label incorporation into stimulated p27, whereas p55 and p18 showed little or no specific dibutyryl cAMP-stimulated labeling. Enzyme substrates T4 and 3,3'5'-triiodothyronine (rT3) specifically blocked p27 labeling, whereas T3 and the competitive 5'D-II inhibitor EMD 21388 (a synthetic flavonoid) were much less effective. Iopanoate, an inhibitor of all deiodinase isozymes, was ineffective in blocking p27 labeling. Inhibition kinetics revealed that iopanoate was a noncompetitive inhibitor of dibutyryl cAMP-stimulated glial cell 5'D-II, suggesting that it interacts at a site distant from the substrate-binding site. These data identify a cAMP-inducible membrane-associated protein (p27) that has many of the properties of 5'D-II.     

Myosin V plays an essential role in the thyroid hormone-dependent endocytosis of type II iodothyronine 5'-deiodinase

In astrocytes, thyroxine modulates type II iodothyronine 5'-deiodinase levels by initiating the binding of the endosomes containing the enzyme to microfilaments, followed by actin-based endocytosis. Myosin V is a molecular motor thought to participate in vesicle trafficking in the brain. In this report, we developed an in vitro actin-binding assay to characterize the thyroid hormone-dependent binding of endocytotic vesicles to microfilaments. Thyroxine and reverse triiodothyronine (EC(50) levels approximately 1 nm) were >100-fold more potent than 3,5,3'-triiodothyronine in initiating vesicle binding to actin fibers in vitro. Thyroxine-dependent vesicle binding was calcium-, magnesium-, and ATP-dependent, suggesting the participation of one or more myosin motors, presumably myosin V. Addition of the myosin V globular tail, lacking the actin-binding head, specifically blocked thyroid hormone-dependent vesicle binding, and direct binding of the myosin V tail to enzyme-containing endosomes was thyroxine-dependent. Progressive NH(2)-terminal deletion of the myosin V tail and domain-specific antibody inhibition studies revealed that the thyroxine-dependent vesicle-tethering domain was localized to the last 21 amino acids of the COOH terminus. These data show that myosin V is responsible for thyroid hormone-dependent binding of primary endosomes to the microfilaments and suggest that this motor mediates the actin-based endocytosis of the type II iodothyronine deiodinase.     

Bioenergetic impact of tissue-specific regulation of iodothyronine deiodinases during nutritional imbalance

The regulation of energy homeostasis by thyroid hormones is unquestionable, and iodothyronine deiodinases are enzymes involved in the metabolic activation or inactivation of these hormones at the cellular level. T3 is produced through the outer ring deiodination of the prohormone T4, which is catalyzed by types 1 and 2 iodothyronine deiodinases, D1 and D2. Conversely, type 3 iodothyronine deiodinase (D3) catalyzes the inner ring deiodination, leading to the inactivation of T4 into reverse triiodothyronine (rT3). Leptin acts as an important modulator of central and peripheral iodothyronine deiodinases, thus regulating cellular availability of T3. Decreased serum leptin during negative energy balance is involved in the down regulation of liver and kidney D1 and BAT D2 activities. Moreover, in high fat diet induced obesity, instead of increased serum T₃ and T₄ secondary to higher circulating leptin and thyrotropin levels, elevated serum rT3 is found, a mechanism that might impair the further increase in oxygen consumption.     

Subcellular distribution of iodothyronine 5′-deiodinase in cerebral cortex from hypothyroid rats

We have examined iodothyronine deiodination in subcellular fractions of cerebral cortex obtained from hypothyroid rats. Enzymatic activities were measured at 37°C in the presence of 20 mM dithiothreitol with ¹²⁵I-labeled T₄ and ¹²⁵I-labeled rT₃ as substrate for 5′-deiodination and ¹³¹I-labeled T₃ as the substrate for the 5-deiodinase. Reaction products were separated by descending paper and/or ion-exchange chromatography. Cerebral cortex subcellular fractions were also characterized by marker enzyme analysis and electron microscopy. Under optimal reaction conditions more than 80% of the 5′-deiodinase was recovered after fractionation. Both 5′-deiodinase and (Na⁺ +K⁺-ATPase showed similar subcellular distributions and were enriched approx. 3-fold in the easily sedimenting membrane fraction and nerve terminal plasma membranes. Crude microsomal membranes (6·10⁶g·min pellet) also showed 2-fold enrichment of these enzymes. Nuclei and isolated mitochondria were devoid of deiodinating activity. T₄ and T₃ 5-deiodinating activity was absent in the easily sedimenting membranes and present but not enriched in particulate fractions containing microsomal membranes. These data suggest that iodothyronine 5′-deiodinase is associated with plasma membrane fractions in the cerebral cortex.     

12-[(5-iodo-4-azido-2-hydroxybenzoyl)amino]dodecanoic acid: biological recognition by cholesterol esterase and acyl-CoA:cholesterol O-acyltransferase

Potential probes of protein cholesterol and fatty acid binding sites, namely, 12-[(5-iodo-4-azido-2-hydroxybenzoyl)amino]dodecanoate (IFA) and its coenzyme A (IFA:CoA) and cholesteryl (IFA:CEA) esters, were synthesized. These radioactive, photoreactive lipid analogues were recognized as substrates and inhibitors of acyl-CoA:cholesterol O-acyltransferase (ACAT) and cholesterol esterase, neutral lipid binding enzymes which are key elements in the regulation of cellular cholesterol metabolism. In the dark, IFA reversibly inhibited cholesteryl [14C]oleate hydrolysis by purified bovine pancreatic cholesterol esterase with an apparent Ki of 150 microM. Cholesterol esterase inhibition by IFA became irreversible after photolysis with UV light and oleic acid (1 mM) provided 50% protection against inactivation. Incubation of homogeneous bovine pancreatic cholesterol esterase with IFA:CEA resulted in its hydrolysis to IFA and cholesterol, indicating recognition of IFA:CEA as a substrate by cholesterol esterase. The coenzyme A ester, IFA:CoA, was a reversible inhibitor of microsomal ACAT activity under dark conditions (apparent Ki = 20 microM), and photolysis resulted in irreversible inhibition of enzyme activity with 87% efficiency. IFA:CoA was also recognized as a substrate by both liver and aortic microsomal ACATs, with resultant synthesis of 125IFA:CEA. IFA and its derivatives, IFA:CEA and IFA:CoA, are thus inhibitors and substrates for cholesterol esterase and ACAT. Biological recognition of these photoaffinity lipid analogues will facilitate the identification and structural analysis of hitherto uncharacterized protein lipid binding sites.     

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.