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ABSTRACT: BACKGROUND: Iodothyronines are charged amino acid derivatives that cannot passively cross a phospholipid bilayer. Transport of thyroid hormones across plasma membranes is mediated by integral membrane proteins belonging to several gene families. These transporters therefore allow or limit access of thyroid hormones into brain. Since thyroid hormones are essential for brain development and cell differentiation, it is expected that genetic deficiency of such transporters would result in neurodevelopmental derangements. SCOPE OF REVIEW: We introduce concepts of thyroid hormone transport into the brain and into brain cells. Important thyroid hormone transmembrane transporters are presented along with their expression patterns in different brain cell types. A focus is placed on monocarboxylate transporter 8 (MCT8) which has been identified as an essential thyroid hormone transporter in humans. Mutations in MCT8 underlie one of the first described X-linked mental retardation syndromes, the Allan-Herndon-Dudley syndrome. MAJOR CONCLUSIONS: Thyroid hormone transporter molecules are expressed in a developmental and cell type-specific pattern. Any thyroid hormone molecule has to cross consecutively the luminal and abluminal membranes of the capillary endothelium, enter astrocytic foot processes, and leave the astrocyte through the plasma membrane to finally cross another plasma membrane on its way towards its target nucleus. GENERAL SIGNIFICANCE: We can expect more transporters being involved in or contributing to in neurodevelopmental or neuropsychiatric disease. Due to their expression in cellular components regulating the hypothalamus-pituitary-thyroid axis, mutations and polymorphisms are expected to impact on negative feedback regulation and hormonal setpoints. This article is part of a Special Issue entitled Thyroid hormone signalling.
Biochimica et Biophysica Acta 08/2012; · 4.66 Impact Factor
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ABSTRACT: Enzymatic 5'- and 5-deiodination are key reactions for local and systemic activation and inactivation of iodothyronines and thyronamines. Expression of the three deiodinase (DIO) isoenzymes is regulated by a number of parameters, including thyroid status, genotype, micronutrient availability, and disease-related signaling. In addition, DIO are potential targets of pharmacological as well as environmentally derived substances, which might affect their enzymatic activity (endocrine disruptors). With the classical DIO activity assay, testing depends on the availability of radioactively labeled substrates (e.g. (125)I-rT(3)) to monitor the release of radioactive iodide. Recently, liquid chromatography-tandem mass spectrometry was described as an alternative method apparently resolving this limitation. However, it has a high demand in technical equipment and analytical routine and is limited in sample number by considerable measuring time. We therefore combined the classical deiodination assay with an easily accessible photometric method taking advantage of the Sandell-Kolthoff reaction for measuring iodide release. In brief, iodine works as a catalyst within this redox reaction between Ce(4+) and As(3+) leading to an acceleration of destaining. Furthermore, the protocol was adapted to minimize handling effort and time consumption. Because this method is not dependent on radioactivity, it expands the substrate spectrum of the classical method. Suitability of this assay was tested with tissue samples from animal experiments (hepatic Dio1 activity in hypo- and hyperthyroid mice) and established DIO inhibitors. As a new but not unexpected finding, the alleged inhibitor iopanoic acid turned out to be a DIO substrate. This finding was confirmed by liquid chromatography-tandem mass spectrometry, and its potential clinical impact requires further studies.
Endocrinology 03/2012; 153(5):2506-13. · 4.46 Impact Factor
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ABSTRACT: We have tested the hypothesis that selenium (Se)-containing antioxidative enzymes protect thyroid epithelial cells from oxidative damage associated with enzymatic production of hydrogen peroxide required for thyroid hormone biosynthesis. Thyroid epithelial cells therefore express antioxidative enzymes, including catalase, peroxiredoxins, thioredoxin reductases, and glutathione peroxidases (GPxs). The latter two enzyme families contain highly active peroxide-degrading enzymes that carry selenocysteine (Sec) in their active centers. Since low Se status has been associated with thyroid disorders, selenoproteins are considered essential for thyroid integrity and function. We have conditionally inactivated selenoprotein biosynthesis in thyrocytes by targeting Sec tRNA.
Constitutive and inducible Cre/loxP-mediated recombination of tRNA([Ser]Sec) drastically reduced activities of selenoenzymes GPx and type I-deiodinase in thyroid extracts. Immunohistochemical staining revealed increased 4-hydroxynonenal and 3-nitro-tyrosine levels consistent with increased oxidative stress. However, gross thyroid morphology remained intact for at least 6 months after recombination. Circulating thyroid hormone levels remained normal in mutant mice, while thyrotropin (TSH) levels were moderately elevated. Challenging mutant mice with low iodine diet increased TSH, but did not lead to destruction of selenoprotein-deficient thyroids.
This is the first report probing the assumed physiological roles of selenoproteins in the thyroid using a genetic loss-of-function approach.
We conclude that selenoproteins protect thyrocytes from oxidative damage and modulate thyroid hormone biosynthesis, but are not essential for thyrocyte survival.
Antioxidants & Redox Signaling 02/2012; 17(6):902-13. · 8.20 Impact Factor
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ABSTRACT: Tyrosine kinase inhibitors (TKI) are used for the treatment of various cancers. Case reports and clinical trials have reported abnormal thyroid function tests (TFT) after treatment with sunitinib, imatinib, sorafenib, dasatinib, and nilotinib. An increased requirement for levothyroxine was reported in thyroidectomized patients during TKI treatment.
We hypothesized that abnormal TFT are compatible with inhibition of thyroid hormone (TH) transporters and subsequently reduced pituitary-TH feedback. Monocarboxylate transporter 8 (MCT8) is a TH transmembrane transporter in brain, pituitary, and other organs. MCT8 mutation leads to abnormal TFT in patients and respective mouse models. We tested whether TKI are able to inhibit MCT8-mediated TH uptake into cells.
Madin-Darby-canine kidney (MDCK1) cells stably expressing human MCT8 were exposed in vitro to TKI at increasing concentrations, and MCT8-mediated [(125)I]T(3) uptake and efflux were measured. The mode of inhibition was determined.
TKI exposure dose-dependently inhibited MCT8-dependent T(3) and T(4) uptake. IC(50) values for sunitinib, imatinib, dasatinib, and bosutinib ranged from 13-38 μm, i.e. similar to the Michaelis-Menten constant K(m) for T(3) and T(4), 4 and 8 μm, respectively. Kinetic experiments revealed a noncompetitive mode of inhibition for all TKI tested.
Partial inhibition by TKI of pituitary or hypothalamic TH feedback may increase TSH or increase the levothyroxine requirement of thyroidectomized patients. It is still possible that other mechanisms contribute to TKI-mediated impairments of TFT, e.g. altered metabolism of TH. Bosutinib was not previously reported to alter TFT.
The Journal of clinical endocrinology and metabolism 01/2012; 97(1):E100-5. · 6.50 Impact Factor
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Johann Ockenga,
Luzia Valentini,
Tatjana Schuetz,
Franziska Wohlgemuth,
Silja Glaeser,
Ajmal Omar,
Esmatollah Kasim,
Daniel duPlessis,
Karen Featherstone,
Julian R Davis,
Uwe J F Tietge,
Thomas Kroencke,
Heike Biebermann, Josef Köhrle,
Georg Brabant
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ABSTRACT: Animal studies implicate a role of bile acids (BA) in thyroid-regulated energy expenditure (EE) via activation of the TGR-5/adenylate cyclase/deiodinase type 2 pathway. Here we investigated these possible associations in humans.
EE, BA, and thyroid hormone status were assessed in 10 healthy subjects and eight patients with liver cirrhosis at baseline and after oral nutrition. In cirrhosis, blood was additionally sampled from the mesenteric vein and the radial artery.
At baseline, BA and EE related positively (r = 0.648, P = 0.048 in healthy subjects; r = 0.833, P = 0.010 in cirrhosis; r = 0.556, P =0.017 in all), with the highest correlation with deoxycholic acid levels. The respiratory quotient associated negatively to baseline BA (all, r = -0.639, P = 0.004). Postprandially, serum TSH decreased in both groups (P < 0.05 each). In cirrhosis, the decrease of TSH after 60 min correlated to the meal-stimulated BA increase (r = -0.762, P = 0.028). To assess the mechanism involved, we studied a single human TSHoma and TαT1 mouse thyrotrope cells. In TSHoma cells, TGR-5 was predominantly expressed cytoplasmically, and in vitro stimulation with BA did not substantially alter cAMP or deiodinase type 2.
Our data support a role of BA in human energy metabolism and in thyroid hormone control. Even though no convincing response to BA was demonstrated in TSHoma and TαT1 cells, the TSH decrease after a nutritional challenge suggests an interaction of BA on the set point of the thyroid axis.
The Journal of clinical endocrinology and metabolism 12/2011; 97(2):535-42. · 6.50 Impact Factor
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Carolin S Hoefig,
Kostja Renko,
Susanne Piehl,
Thomas S Scanlan,
Mariarita Bertoldi,
Thomas Opladen,
Georg Friedrich Hoffmann,
Jeannette Klein,
Oliver Blankenstein,
Ulrich Schweizer, Josef Köhrle
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ABSTRACT: Thyronamines (TAM), recently described endogenous signaling molecules, exert metabolic and pharmacological actions partly opposing those of the thyromimetic hormone T(3). TAM biosynthesis from thyroid hormone (TH) precursors requires decarboxylation of the L-alanine side chain and several deiodination steps to convert e.g. L-thyroxine (T(4)) into the most potent 3-T(1)AM. Aromatic L-amino acid decarboxylase (AADC) was proposed to mediate TAM biosynthesis via decarboxylation of TH. This hypothesis was tested by incubating recombinant human AADC, which actively catalyzes dopamine production from DOPA, with several TH. Under all reaction conditions tested, AADC failed to catalyze TH decarboxylation, thus challenging the initial hypothesis. These in vitro observations are supported by detection of 3-T(1)AM in plasma of patients with AADC-deficiency at levels (46 ± 18 nM, n=4) similar to those of healthy controls. Therefore, we propose that the enzymatic decarboxylation needed to form TAM from TH is catalyzed by another unique, perhaps TH-specific, decarboxylase.
Molecular and Cellular Endocrinology 10/2011; 349(2):195-201. · 4.19 Impact Factor
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Eva K Wirth,
Sien-Yi Sheu,
Jazmin Chiu-Ugalde,
Remy Sapin,
Marc O Klein,
Ilona Mossbrugger,
Leticia Quintanilla-Martinez,
Martin Hrabĕ de Angelis,
Heiko Krude,
Thomas Riebel,
Karin Rothe, Josef Köhrle,
Kurt W Schmid,
Ulrich Schweizer,
Annette Grüters
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ABSTRACT: Thyroid hormone transport across the plasma membrane depends on transmembrane transport proteins, including monocarboxylate transporter 8 (MCT8). Mutations in MCT8 (or SLC16A2) lead to a severe form of X-linked psychomotor retardation, which is characterised by elevated plasma triiodothyronine (T(3)) and low/normal thyroxine (T(4)). MCT8 contributes to hormone release from the thyroid gland.
To characterise the potential impact of MCT8-deficiency on thyroid morphology in a patient and in Mct8-deficient mice.
Thyroid morphology in a patient carrying the A224V mutation was followed by ultrasound imaging for over 10 years. After thyroidectomy, a histopathological analysis was carried out. The findings were compared with histological analyses of mouse thyroids from the Mct8(-/y) model.
We show that an inactivating mutation in MCT8 leads to a unique, progressive thyroid follicular pathology in a patient. After thyroidectomy, histological analysis revealed gross morphological changes, including several hyperplastic nodules, microfollicular areas with stromal fibrosis and a small focus of microfollicular structures with nuclear features reminiscent of papillary thyroid carcinoma (PTC). These findings are supported by an Mct8-null mouse model in which we found massive papillary hyperplasia in 6- to 12-month-old mice and nuclear features consistent with PTC in almost 2-year-old animals. After complete thyroidectomy and substitution with levothyroxine (l-T(4)), the preoperative, inadequately low T(4) and free T(4) remained, while increasing the l-T(4) dosage led to T(3) serum concentrations above the normal range.
Our results implicate peripheral deiodination in the peculiar hormonal constellation of MCT8-deficient patients. Other MCT8-deficient patients should be closely monitored for potential thyroid abnormalities.
European Journal of Endocrinology 08/2011; 165(4):555-61. · 3.42 Impact Factor
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ABSTRACT: Patients presenting to emergency departments (ED) with nonspecific complaints (NSCs) such as "not feeling well,"feeling weak,"being tired,"general deterioration," or other similar chief complaints that do not have a readily identifiable probable etiology are a common patient group at risk for adverse outcomes. Certain biomarkers, which have not yet been tested for prognostic value when applied to ED patients with NSCs, have emerged as useful tools for predicting prognosis in patients with a variety of diseases. This study tested the hypothesis that two of these novel markers, copeptin (a C-terminal portion of provasopressin) and/or peroxiredoxin-4 (Prx4), an enzyme that degrades hydrogen peroxide, singly or together are helpful in predicting death in the near term among patients presenting to the ED with NSCs.
The Basel Non-specific Complaints (BANC) study is a delayed type cross-sectional diagnostic study with a prospective 30-day follow-up. ED patients with NSCs were consecutively enrolled. Patients with vital parameters out of the normal range were excluded. The primary endpoint of this study was the predictive value of copeptin and Prx4 for 30-day mortality in patients with NSCs. Measurement of both copeptin and Prx4 was performed in serum samples with sandwich immunoluminometric assays.
On follow-up at 30 days after ED presentation, 28 of 438 patients with NSC had died. Copeptin and Prx4 concentrations were significantly higher in nonsurvivors than in survivors (Kruskal-Wallis test, p = 0.0001 and p < 0.0001, respectively). In univariate models, Prx4 (likelihood ratio [LR] χ(2) = 22.24, p < 0.00001, concordance index [C-index] = 0.749) and copeptin (LR χ(2) = 16.98, p = 0.00004, C-index = 0.724) were both predictive of 30-day mortality, and elevated levels were associated with an increased mortality. The bivariable model, which included both Prx4 and copeptin (LR χ(2) = 28.22, p < 0.00001, C-index = 0.783), allows a significantly better prediction than the univariate Prx4 (p = 0.00025) and copeptin models (p = 0.00099), respectively. Both biomarkers provided independent and additional information to clinical risk scores (Katz Activities of Daily Living [ADL] and Charlson Comorbidity Index [CCI], all p < 0.0005).
Copeptin and Prx4 are new prognostic markers in patients presenting to the ED with NSCs. Copeptin and Prx4 might be valuable tools for risk stratification and decision-making in this patient group.
Academic Emergency Medicine 08/2011; 18(8):851-9. · 1.86 Impact Factor
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ABSTRACT: LAT2 (system L amino acid transporter 2) is composed of the subunits Slc7a8/Lat2 and Slc3a2/4F2hc. This transporter is highly expressed along the basolateral membranes of absorptive epithelia in kidney and small intestine, but is also abundant in the brain. Lat2 is an energy-independent exchanger of neutral amino acids, and was shown to transport thyroid hormones. We report in the present paper that targeted inactivation of Slc7a8 leads to increased urinary loss of small neutral amino acids. Development and growth of Slc7a8(-/-) mice appears normal, suggesting functional compensation of neutral amino acid transport by alternative transporters in kidney, intestine and placenta. Movement co-ordination is slightly impaired in mutant mice, although cerebellar development and structure remained inconspicuous. Circulating thyroid hormones, thyrotropin and thyroid hormone-responsive genes remained unchanged in Slc7a8(-/-) mice, possibly because of functional compensation by the thyroid hormone transporter Mct8 (monocarboxylate transporter 8), which is co-expressed in many cell types. The reason for the mild neurological phenotype remains unresolved.
Biochemical Journal 07/2011; 439(2):249-55. · 4.90 Impact Factor
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ABSTRACT: Thyronamines are thyronergic metabolites of thyroid hormones. Lack of reliable and sensitive detection methods for endogenous 3-iodothyronamine (3-T(1)AM) has so far hampered progress in understanding their physiological action and role in endocrine homeostasis or pathophysiology of diseases.
We characterized newly generated mouse monoclonal 3-T(1)AM antibodies and established a monoclonal antibody-based chemiluminescence immunoassay as a powerful tool for monitoring 3-T(1)AM levels in investigations addressing altered serum profiles and potential sites of origin and action of 3-T(1)AM in humans.
Our exploratory study on 3-T(1)AM serum levels in humans measured 3-T(1)AM concentrations in comparison with thyroid hormones.
Thirteen adult healthy subjects, 10 patients with pituitary insufficiency, and 105 thyroid cancer patients participated. Interventions: Interventions included l-T(4) withdrawal in patients with pituitary insufficiency as well as TSH-suppressive T(4) substitution in thyroid cancer patients.
3-T(1)AM was reliably quantified in human serum and stable after storage at room temperature and 4 C overnight as well as after four freeze-thaw cycles. The median serum concentration in healthy subjects was 66 ± 26 nm. 3-T(1)AM was also detected in T(4)-substituted thyroid cancer patients. Although free T(4) and T(3) significantly decreased during T(4) withdrawal, 3-T(1)AM levels remained constant for 6 d.
Because higher 3-T(1)AM levels are detectable in T(4)-substituted thyroid cancer patients after thyroidectomy/radioiodine treatment compared with healthy controls, we concluded that 3-T(1)AM is mainly produced by extrathyroidal tissues. The serum profile during T(4) withdrawal suggests either a long half-life or persisting 3-T(1)AM release into serum from intracellular thyroid hormone precursors or stores.
The Journal of clinical endocrinology and metabolism 06/2011; 96(6):1864-72. · 6.50 Impact Factor
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ABSTRACT: Cellular thyroid hormone uptake and efflux are mediated by transmembrane transport proteins. One of these, monocarboxylate transporter 8 (MCT8) is mutated in Allan-Herndon-Dudley syndrome, a severe mental retardation associated with abnormal thyroid hormone constellations. Since mice deficient in Mct8 exhibit a milder neurological phenotype than patients, we hypothesized that alternative thyroid hormone transporters may compensate in murine brain cells for the lack of Mct8. Using qPCR, Western Blot, and immunocytochemistry, we investigated the expression of three different thyroid hormone transporters, i.e., Mct8 and L-type amino acid transporters Lat1 and Lat2, in mouse brain. All three thyroid hormone transporters are expressed from corticogenesis and peak around birth. Primary cultures of neurons and astrocytes express Mct8, Lat1, and Lat2. Microglia specifically expresses Mct10 and Slco4a1 in addition to high levels of Lat2 mRNA and protein. As in vivo, a brain microvascular endothelial cell line expressed Mct8 and Lat1. 158N, an oligodendroglial cell line expressed Mct8 protein, consistent with delayed myelination in MCT8-deficient patients. Functional T(3)- and T(4)-transport assays into primary astrocytes showed K(M) values of 4.2 and 3.7 μM for T(3) and T(4). Pharmacological inhibition of L-type amino acid transporters by BCH and genetic inactivation of Lat2 reduced astrocytic T(3) uptake to the same extent. BSP, a broad spectrum inhibitor, including Mct8, reduced T(3) uptake further suggesting the cooperative activity of several T(3) transporters in astrocytes.
Glia 03/2011; 59(3):463-71. · 4.82 Impact Factor
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ABSTRACT: The objective was to study the associations between serum selenium concentration and thyroid volume, as well as the association between serum selenium concentration and risk for an enlarged thyroid gland in an area with mild iodine deficiency before and after iodine fortification was introduced. Another objective was to examine the association between serum selenium concentration and prevalence of thyroid nodules.
Cross-sectional study.
We studied participants of two similar cross-sectional studies carried out before (1997-1998, n=405) and after (2004-2005, n=400) introduction of iodine fortification. Serum selenium concentration and urinary iodine were measured, and the thyroid gland was examined by ultrasonography in the same subjects. Associations between serum selenium concentration and thyroid parameters were examined in multiple linear regression models or logistic regression models.
Serum selenium concentration was found to be significantly, negatively associated with thyroid volume (P=0.006), and a low selenium status significantly increased the risk for thyroid enlargement (P=0.007). Furthermore, low serum selenium status had a tendency to increase the risk for development of multiple nodules (P=0.087).
Low serum selenium concentration was associated with a larger thyroid volume and a higher prevalence of thyroid enlargement.
European Journal of Endocrinology 01/2011; 164(4):585-90. · 3.42 Impact Factor
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ABSTRACT: Oxidative stress, a situation with increased reactive oxygen species production and/or decreased antioxidant defense mechanisms, is evident in the pathogenesis of sepsis. Peroxiredoxin 4 (Prx4) is a hydrogen peroxide degrading peroxidase recently found circulating in blood of septic patients and potentially reflecting an antioxidant system in imbalance. We studied Prx4 serum levels of 79 consecutively enrolled medical intensive care unit patients. The diagnostic and prognostic performance of Prx4 was compared with other biomarkers, the APACHE II score and the SOFA score. Median Prx4 serum levels gradually increased with disease severity in patients classified on admission as having systemic immune response syndrome (2.32 arbitrary [arb.] U/L), sepsis (5.02 arb. U/L), severe sepsis (11.7 arb. U/L), or septic shock (11.4 arb. U/L). A positive correlation was found with the severity score Acute Physiological and Chronic Health Evaluation II (r = 0.27, P < 0.05) and the organ failure score Sequential Organ Failure Assessment (r = 0.55, P < 0.0001). Peroxiredoxin 4 correlated with the sepsis marker procalcitonin (r = 0.61, P < 0.0001), the inflammatory markers C-reactive protein (r = 0.65, P < 0.0001) and interleukin 6 (r = 0.62, P < 0.0001), and antioxidant blood compounds total bilirubin (r = 0.37, P < 0.001) and albumin (r = -0.54, P < 0.0001). Peroxiredoxin 4 distinguished noninfectious from infectious inflammatory response syndrome with an area under the receiver operating characteristic (ROC) curve of 0.82. [corrected] High Prx4 serum levels were associated with a poor prognosis of septic patients and revealed an area under the ROC curve of 0.76 in prediction of in-hospital mortality. In this study, elevated serum levels of the antioxidant Prx4 were associated with an increased disease severity and adverse outcome of critically ill patients with sepsis. Peroxiredoxin 4 may therefore be a helpful new biomarker for diagnosing, monitoring, and risk assessing these patients. The pathophysiological mechanisms behind the observed increase remain to be elucidated.
Shock (Augusta, Ga.) 01/2011; 35(5):460-5. · 2.87 Impact Factor
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ABSTRACT: Trace amine-associated receptors (TAAR) are rhodopsin-like G-protein-coupled receptors (GPCR). TAAR are involved in modulation of neuronal, cardiac and vascular functions and they are potentially linked with neurological disorders like schizophrenia and Parkinson's disease. Subtype TAAR1, the best characterized TAAR so far, is promiscuous for a wide set of ligands and is activated by trace amines tyramine (TYR), phenylethylamine (PEA), octopamine (OA), but also by thyronamines, dopamine, and psycho-active drugs. Unfortunately, effects of trace amines on signaling of the two homologous β-adrenergic receptors 1 (ADRB1) and 2 (ADRB2) have not been clarified yet in detail. We, therefore, tested TAAR1 agonists TYR, PEA and OA regarding their effects on ADRB1/2 signaling by co-stimulation studies. Surprisingly, trace amines TYR and PEA are partial allosteric antagonists at ADRB1/2, whereas OA is a partial orthosteric ADRB2-antagonist and ADRB1-agonist. To specify molecular reasons for TAAR1 ligand promiscuity and for observed differences in signaling effects on particular aminergic receptors we compared TAAR, tyramine (TAR) octopamine (OAR), ADRB1/2 and dopamine receptors at the structural level. We found especially for TAAR1 that the remarkable ligand promiscuity is likely based on high amino acid similarity in the ligand-binding region compared with further aminergic receptors. On the other hand few TAAR specific properties in the ligand-binding site might determine differences in ligand-induced effects compared to ADRB1/2. Taken together, this study points to molecular details of TAAR1-ligand promiscuity and identified specific trace amines as allosteric or orthosteric ligands of particular β-adrenergic receptor subtypes.
PLoS ONE 01/2011; 6(10):e27073. · 4.09 Impact Factor
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ABSTRACT: The monocarboxylate transporter 8 (MCT8) is a member of the major facilitator superfamily (MFS) and transports specificly iodothyronines. MCT8 mutations are the underlying cause of a syndrome of severe X-linked psychomotor retardation known as the Allan-Herndon-Dudley syndrome. This syndrome is characterized by abnormally high T3, low/normal T4 serum levels and slightly elevated serum TSH. To date, more than 25 pathogenic mutations in hMCT8 are known and they are valuable indicators of important regions for structural and functional MCT8 properties.
We designed a structural human MCT8 model and studied reported pathogenic missense mutations with focus on the estimation of those amino acid positions which are probably sensitive for substrate transport. Furthermore, assuming similarities between determinants of T3 binding observed in the published crystal structure of the thyroid hormone receptor beta occupied by its ligand T3 and the structural MCT8 model, we explore potential T3 binding sites in the MCT8 substrate channel cavity.
We found that all known pathogenic missense mutations are located exclusively in the transmembrane helices and to a high degree at conserved residues among the MCT family. Furthermore, mutations either of or to prolines/glycines are located mainly at helices 9-12 and are expected to cause steric clashes or structural misfolding. In contrast, several other mutations are close to the potential substrate channel and affected amino acids are likely involved in the switching mechanism between different transporter conformations. Finally, three potential substrate binding sites are predicted for MCT8.
Naturally occurring mutations of MCT8 provide molecular insights into protein regions important for protein folding, substrate binding and the switching mechanism during substrate transport. Future studies guided by this information should help to clarify structure-function relationships at MCT8 which may bear broader relevance for other members of the MCT family. This includes decoding of the complete set of transport-sensitive residue positions and description of structural re-arrangements during transport.
Thyroid Research 01/2011; 4 Suppl 1:S4.
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ABSTRACT: The essential micronutrient selenium (Se) exerts its biological effects mainly through enzymatically active selenoproteins. Their biosynthesis depends on the 21st proteinogenic amino acid selenocysteine and thus on dietary Se supply. Hepatically derived selenoprotein P (SEPP) is the central selenoprotein in blood controlling Se transport and distribution. Kidney-derived extracellular glutathione peroxidase is another relevant serum selenoprotein depending on SEPP for biosynthesis. Therefore, secretion of SEPP by hepatocytes is crucial to convert nutritional sources into serum Se, supporting Se status and selenoprotein biosynthesis in other tissues. In order to compare the bioactivity of 10 different selenocompounds, their dose-dependent toxicities and nutritional qualities to support SEPP and glutathione peroxidase biosynthesis were determined in a murine and two human liver cell lines. Characteristic dose- and time-dependent effects on viability and SEPP production were observed. Incubations with 100 nM sodium selenite, l- or dl-selenocystine, selenodiglutathione or selenomethyl-selenocysteine increased SEPP concentrations in the culture medium up to 6.5-fold over control after 72 h. In comparison, sodium selenate, l- or dl-selenomethionine or methylseleninic acid was less effective and increased SEPP by 2.5-fold under these conditions. As expected, ebselen did not increase selenoprotein production, supporting its classification as a stable selenocompound. Methylseleninic acid, l-selenocystine, selenodiglutathione or selenite induced cell death in micromolar concentrations, whereas selenomethionine or ebselen was not toxic within the concentration range tested. Our results indicate that hepatic selenoprotein production and toxicity of selenocompounds do not correlate with and rather represent compound-specific properties. The favourable profile of selenomethylselenocysteine warrants its consideration as a promising option for supplementation purposes.
The Journal of nutritional biochemistry 12/2010; 22(10):945-55. · 4.29 Impact Factor
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ABSTRACT: Distribution of selenium (Se) within the mammalian body is mediated by SePP (selenoprotein P), an Se-rich glycoprotein secreted by hepatocytes. Genetic and biochemical evidence indicate that the endocytic receptors ApoER2 (apolipoprotein E receptor 2) and megalin mediate tissue-specific SePP uptake. In the present study megalin-mutant mice were fed on diets containing adequate (0.15 p.p.m.) or low (0.08 p.p.m.) Se content and were analysed for tissue and plasma Se levels, cellular GPx (glutathione peroxidase) activities and protein expression patterns. Megalin-mutant mice displayed increased urinary Se loss, which correlated with SePP excretion in their urine. Accordingly, serum Se and SePP levels were significantly reduced in megalin-mutant mice, reaching marginal levels on the low-Se diet. Moreover, kidney Se content and expression of renal selenoproteins were accordingly reduced, as was SePP internalization along the proximal tubule epithelium. Although GPx4 expression was not altered in testis, Se and GPx activity in liver and brain were significantly reduced. When fed on a low-Se diet, megalin-mutant mice developed impaired movement co-ordination, but no astrogliosis. These findings suggest that megalin prevents urinary SePP loss and participates in brain Se/SePP uptake.
Biochemical Journal 10/2010; 431(1):103-11. · 4.90 Impact Factor
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ABSTRACT: We describe the validation of a novel assay for the measurement of peroxiredoxin 4 (Prx4), a potentially secreted antioxidant peroxidase, in human serum.
A sandwich immunoluminometric assay (ILMA) was set up applying monoclonal antibodies against the amino-terminus of human Prx4. Prx4 levels have been determined in serum of healthy individuals and patients with sepsis and have been correlated to the clinically established sepsis marker procalcitonin (PCT).
The sandwich ILMA detected Prx4 in a range between 0.5 and 128 arbitrary (arb.) U/L and had a functional assay sensitivity of 0.51 arb.U/L. Serum Prx4 was stable for at least 72 h at 4 degrees C and 21 degrees C and circulated in a complex of about 330 kDa as characterized by size-exclusion chromatography. Patients with sepsis (n=45; median, 7.7 arb.U/L) showed significantly higher Prx4 serum levels (P<0.0001) than healthy controls (n=274; median, 0.71 arb.U/L). Serum Prx4 was positively correlated to PCT (r=0.63, P<0.0001).
The newly developed assay reliably detected Prx4 in human serum of healthy and critically ill subjects. Elevated Prx4 in serum of patients with various diseases might serve as a biomarker reflecting increased oxidative stress.
Clinica chimica acta; international journal of clinical chemistry 09/2010; 411(17-18):1258-63. · 2.54 Impact Factor
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ABSTRACT: Monocarboxylate transporter 8 (MCT8, SLC16A2) is a thyroid hormone (TH) transmembrane transport protein mutated in Allan-Herndon-Dudley syndrome, a severe X-linked psychomotor retardation. The neurological and endocrine phenotypes of patients deficient in MCT8 function underscore the physiological significance of carrier-mediated TH transmembrane transport. MCT8 belongs to the major facilitator superfamily of 12 transmembrane-spanning proteins and mediates energy-independent bidirectional transport of iodothyronines across the plasma membrane. Structural information is lacking for all TH transmembrane transporters. To gain insight into structure-function relations in TH transport, we chose human MCT8 as a paradigm. We systematically performed conventional and liquid chromatography-tandem mass spectrometry-based uptake measurements into MCT8-transfected cells using a large number of compounds structurally related to iodothyronines. We found that human MCT8 is specific for L-iodothyronines and requires at least one iodine atom per aromatic ring. Neither thyronamines, decarboxylated metabolites of iodothyronines, nor triiodothyroacetic acid and tetraiodothyroacetic acid, TH derivatives lacking both chiral center and amino group, are substrates for MCT8. The polyphenolic flavonoids naringenin and F21388, potent competitors for TH binding at transthyretin, did not inhibit T(3) transport, suggesting that MCT8 can discriminate its ligand better than transthyretin. Bioinformatic studies and a first molecular homology model of MCT8 suggested amino acids potentially involved in substrate interaction. Indeed, alanine mutation of either Arg(445) (helix 8) or Asp(498) (helix 10) abrogated T(3) transport activity of MCT8, supporting their predicted role in substrate recognition. The MCT8 model allows us to rationalize potential interactions of amino acids including those mutated in patients with Allan-Herndon-Dudley syndrome.
Journal of Biological Chemistry 09/2010; 285(36):28054-63. · 4.77 Impact Factor
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ABSTRACT: Our previous studies demonstrated that retinoic acid (RA)-induced reduction of both, the key glycolytic enzyme ENO1 and proliferation-promoting c-Myc, resulted in decreased vitality and invasiveness of the follicular thyroid carcinoma cell lines FTC-133 and FTC-238. By employing two-dimensional electrophoresis and mass spectrometry, we identified proteins affected by RA treatment. In addition to previously reported decrease in ENO1 expression, we found that RA led to significantly reduced levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), pyruvate kinase isoenzymes M1/M2 (PKM1/M2), peptidyl-prolyl cis-trans isomerase A (PPIA), transketolase (TKT), annexin A2 (ANXA2), glutathione S-transferase P (GSTP1) and peroxiredoxin 2 (PRDX2) as compared to untreated control. The same proteins investigated on thyroid tissues were found to be significantly up-regulated in follicular, papillary and undifferentiated thyroid carcinomas when compared with goiter and adenoma tissues. These findings identify new target proteins for RA-mediated anti-tumor and re-differentiation therapies and provide novel insights into treatments for thyroid carcinoma.
Molecular and Cellular Endocrinology 08/2010; 325(1-2):110-7. · 4.19 Impact Factor
