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Thyroid : Official Journal of the... May 2024Monocarboxylate transporter 8 (MCT8) is the most specific thyroid hormone transporter identified to date, deficiency of which has been associated with severe...
Monocarboxylate transporter 8 (MCT8) is the most specific thyroid hormone transporter identified to date, deficiency of which has been associated with severe intellectual and motor disability and abnormal serum thyroid function tests. However, it is presently unknown if MCT8, similar to other thyroid hormone transporters, also accepts additional substrates, and if disruption of their transport may contribute to the observed phenotype. In this study, we aimed to identify such substrates by applying liquid chromatography-mass spectrometry-based metabolome analysis in lysates of control and MCT8-overexpressing oocytes. A subset of identified candidate substrates were validated by direct transport studies in transiently transfected COS-1 cells and human fibroblasts, which endogenously express MCT8. Moreover, transport characteristics were determined, including transport saturation and cis-inhibition potency of thyroid hormone transport. Metabolome analysis identified 21 m/z ratios, corresponding to 87 candidate metabolites, with a 2.0-times differential abundance in MCT8-injected oocytes compared with controls. These metabolites included 3,5-diiodotyrosine (DIT) and several amino acids, including glutamate and glutamine. In accordance, MCT8-expressing COS-1 cells had 2.2-times lower intracellular accumulation of [I]-DIT compared with control cells. This effect was largely blocked in the presence of 3,3',5-triiodothyronine (T3) (IC: 2.5 ± 1.5 µM) or thyroxine (T4) (IC: 5.8 ± 1.3 µM). Conversely, increasing concentrations of DIT enhanced the accumulation of T3 and T4. The MCT8-specific inhibitor silychristin increased the intracellular accumulation of DIT in human fibroblasts. COS-1 cells expressing MCT8 also exhibited a 50% reduction in intracellular accumulation of [I]-3-monoiodotyrosine (MIT). In contrast, COS-1 cells expressing MCT8 did not alter the intracellular accumulation of [H]-glutamate or [H]-glutamine. However, studies in human fibroblasts showed a 1.5-1.9 times higher glutamate uptake in control fibroblasts compared with fibroblasts derived from patients with MCT8 deficiency, which was not affected in the presence of silychristin. Taken together, our results suggest that the iodotyrosines DIT and MIT can be exported by MCT8. MIT and DIT interfere with MCT8-mediated transport of thyroid hormone and . Future studies should elucidate if MCT8, being highly expressed in thyroidal follicular cells, also transports iodotyrosines .
PubMed: 38661522
DOI: 10.1089/thy.2023.0551 -
Thyroid : Official Journal of the... Jun 2023Iodine is required for the synthesis of thyroid hormone (TH), but its natural availability is limited. Dehalogenase1 (Dehal1) recycles iodine from mono- and...
Iodine is required for the synthesis of thyroid hormone (TH), but its natural availability is limited. Dehalogenase1 (Dehal1) recycles iodine from mono- and diiodotyrosines (MIT, DIT) to sustain TH synthesis when iodine supplies are scarce, but its role in the dynamics of storage and conservation of iodine is unknown. -knockout (KO) mice were generated by gene trapping. The timing of expression and distribution was investigated by X-Gal staining and immunofluorescence using recombinant Dehal1-beta-galactosidase protein produced in fetuses and adult mice. Adult KO and wild-type () animals were fed normal and iodine-deficient diets for 1 month, and plasma, urine, and tissues were isolated for analyses. TH status was monitored, including thyroxine, triiodothyronine, MIT, DIT, and urinary iodine concentration (UIC) using a novel liquid chromatography with tandem mass spectrometry method and the Sandell-Kolthoff (S-K) technique throughout the experimental period. Dehal1 is highly expressed in the thyroid and is also present in the kidneys, liver, and, unexpectedly, the choroid plexus. transcription of was induced by iodine deficiency only in the thyroid tissue. Under normal iodine intake, KO mice were euthyroid, but they showed negative iodine balance due to a continuous loss of iodotyrosines in the urine. Counterintuitively, the UIC of KO mice is twofold higher than that of mice, indicating that S-K measures both inorganic and organic iodine. Under iodine restriction, KO mice rapidly develop profound hypothyroidism, while mice remain euthyroid, suggesting reduced retention of iodine in the thyroids of KO mice. Urinary and plasma iodotyrosines were continually elevated throughout the life cycles of KO mice, including the neonatal period, when pups were still euthyroid. Plasma and urine iodotyrosine elevation occurs in Dehal1-deficient mice throughout life. Therefore, measurement of iodotyrosines predicts an eventual iodine shortage and development of hypothyroidism in the preclinical phase. The prompt establishment of hypothyroidism upon the start of iodine restriction suggests that KO mice have low iodine reserves in their thyroid glands, pointing to defective capacity for iodine storage.
Topics: Mice; Animals; Monoiodotyrosine; Mice, Knockout; Iodide Peroxidase; Hypothyroidism; Biomarkers; Thyroxine; Iodine
PubMed: 36879468
DOI: 10.1089/thy.2022.0537 -
Thyroid : Official Journal of the... Feb 2023Congenital hypothyroidism due to defects in iodotyrosine deiodinase has variable phenotypes and can present as hypothyroid or with normal thyroid testing. Whole exome...
Congenital hypothyroidism due to defects in iodotyrosine deiodinase has variable phenotypes and can present as hypothyroid or with normal thyroid testing. Whole exome sequencing was performed in individuals from two families originating from different regions of Sudan. Mass spectrometry of urine and serum iodotyrosines was performed on subjects from both families. A novel iodotyrosine deiodinase (IYD) mutation (c.835C>T; R279C) was identified in individuals from two Sudanese families inherited as autosomal recessive. The mutation was identified by multiple in silica analyses to likely be detrimental. Serum and urine monoiodotyrosine (MIT) and diiodotyrosine (DIT) were markedly elevated in the homozygous subjects. Measurement of serum and urine DIT and MIT was more sensitive than that of urine iodine or serum thyroid function tests to determine the effect of the IYD mutation.
Topics: Humans; Congenital Hypothyroidism; Diiodotyrosine; Iodide Peroxidase; Monoiodotyrosine; Mutation
PubMed: 36633921
DOI: 10.1089/thy.2022.0492 -
Molecular and Cellular Endocrinology Dec 2022Thyroid peroxidase (TPO) is a membrane-bound glycoprotein located at the apical side of the thyroid follicular cells that catalyzes both iodination and coupling of... (Review)
Review
Thyroid peroxidase (TPO) is a membrane-bound glycoprotein located at the apical side of the thyroid follicular cells that catalyzes both iodination and coupling of iodotyrosine residues within the thyroglobulin molecule, leading to the synthesis of thyroid hormone. Variants in TPO cause congenital hypothyroidism (CH) by iodide organification defect and are commonly inherited in an autosomal recessive fashion. In the present work, we report a detailed population analysis and bioinformatic prediction of the TPO variants indexed in the Genome Aggregation Database (gnomAD) v2.1.1. The proportion of missense cysteine variants and nonsense, frameshift, and splice acceptor/donor variants were analyzed in each ethnic group (European (Non-Finnish), European (Finnish), African/African Americans, Latino/Admixed American, East Asian, South Asian, Ashkenazi Jewish, Other). The results showed a clear predominance of frameshift variants in the East Asian (82%) and European (Finnish) (75%) population, whereas the splice site variants predominate in African/African Americans (99.46%), Other (96%), Latino/Admixed American (94%), South Asian (86%), European (Non-Finnish) (56%) and Ashkenazi Jewish (56%) populations. The analysis of the distribution of the variants indexed in gnomAD v2.1.1 database revealed that most missense variants identified in the An peroxidase domain map in exon 8, followed by exons 11, 7 and 9, and finally in descending order by exons 10, 6, 12 and 5. In total, 183 novel TPO variants were described (13 missense cysteine's variants, 158 missense variants involving the An peroxidase domain and 12 splicing acceptor or donor sites variants) which were not reported in the literature and that would have deleterious effects on prediction programs. In the gnomAD v2.1.1 population, the estimated prevalence of heterozygous carriers of the potentially damaging variants was 1:77. In conclusion, we provide an updated and curated reference source of new TPO variants for application in clinical diagnosis and genetic counseling. Also, this work contributes to elucidating the molecular basis of CH associated with TPO defects.
Topics: Humans; Thyroglobulin; Iodide Peroxidase; Monoiodotyrosine; Iodides; Computational Biology; Cysteine; Congenital Hypothyroidism; Thyroid Hormones; Mutation; Peroxidases; Algorithms
PubMed: 35995307
DOI: 10.1016/j.mce.2022.111748 -
The Analyst Mar 2022High levels of 3-mono- and 3,5-diiodotyrosine (MIT and DIT, respectively) in urine have been related to iodotyrosine dehalogenase 1 deficiency, a type of congenital...
High levels of 3-mono- and 3,5-diiodotyrosine (MIT and DIT, respectively) in urine have been related to iodotyrosine dehalogenase 1 deficiency, a type of congenital hypothyroidism. However, the determination of MIT and DIT in urine is not included in newborn screening programs performed in clinical laboratories to detect inborn errors of metabolism. We report here on the development of an analytical method for the determination of MIT and DIT in newborn urine and dried urine spots (DUS) by Liquid Chromatography Isotope Dilution tandem Mass Spectrometry (LC-IDMSMS). The development included the synthesis of N-monoiodotyrosine and C-diiodotyrosine through the iodination of N-tyrosine and C-tyrosine, respectively, using bis(pyridine)iodonium(I) tetrafluoroborate (IPyBF). Both labelled analogues were added at the beginning of the sample preparation procedure and used to develop both single- and double-spike LC-IDMS methods for the determination of MIT and DIT. The developed double spike methodology was able to quantify and correct possible MIT ↔ DIT interconversions throughout the sample preparation, which was observed for concentrated urine samples but not for DUS. Suppression matrix effects on the absolute signals of MIT and DIT were observed in urine samples but did not affect the IDMS results as recoveries on urine samples at different dilution factors could be considered quantitative. Method detection limits were 0.018 and 0.046 ng g (limits of quantification 0.06 and 0.15 ng g) by single-spike IDMS, for MIT and DIT, respectively, in the analysis of urine samples and 0.07 and 0.05 ng g (limits of quantification 0.23 and 0.17 ng g) for MIT and DIT, respectively, in the analysis of DUS. No significant differences were obtained for MIT concentrations in the analysis of the same newborn samples stored as liquid urine or DUS when the results were corrected for the creatinine content. Finally, 36 DUS samples from healthy newborns were analyzed and MIT was detected in all samples at low ng mgcreatinine levels.
Topics: Chromatography, Liquid; Diiodotyrosine; Humans; Infant, Newborn; Iodide Peroxidase; Monoiodotyrosine; Tandem Mass Spectrometry
PubMed: 35262098
DOI: 10.1039/d1an02203b -
European Journal of Endocrinology Jan 2022Conventional diagnostic methods are limited in their ability to differentiate destructive thyroiditis from Graves' disease. We hypothesised that serum diiodotyrosine...
OBJECTIVE
Conventional diagnostic methods are limited in their ability to differentiate destructive thyroiditis from Graves' disease. We hypothesised that serum diiodotyrosine (DIT) and monoiodotyrosine (MIT) levels could be biomarkers for differentiating destructive thyroiditis from Graves' disease.
DESIGN
Patients with destructive thyroiditis (n = 13) and Graves' disease (n = 22) were enrolled in this cross-sectional study.
METHODS
We assayed the serum DIT and MIT levels using liquid chromatography-tandem mass spectrometry. A receiver operating characteristic (ROC) curve analysis was used to determine the sensitivity and specificity of the serum DIT and MIT levels as biomarkers for differentiating destructive thyroiditis from Graves' disease.
RESULTS
The serum DIT and MIT levels were significantly higher in patients with destructive thyroiditis than in those with Graves' disease. The ROC curve analysis showed that the serum DIT levels (≥359.9 pg/mL) differentiated destructive thyroiditis from Graves' disease, significantly, with 100.0% sensitivity and 95.5% specificity (P < 0.001). The diagnostic accuracy of the serum MIT levels (≥119.4 pg/mL) was not as high as that of the serum DIT levels (sensitivity, 84.6%; specificity, 77.3%; P = 0.001).
CONCLUSIONS
The serum DIT levels may serve as a novel diagnostic biomarker for differentiating destructive thyroiditis from Graves' disease.
Topics: Adult; Aged; Biomarkers; Cross-Sectional Studies; Diagnosis, Differential; Diiodotyrosine; Female; Graves Disease; Humans; Immunoglobulins, Thyroid-Stimulating; Male; Middle Aged; Monoiodotyrosine; ROC Curve; Sensitivity and Specificity; Thyroiditis; Thyrotoxicosis; Thyrotropin; Thyroxine
PubMed: 34874894
DOI: 10.1530/EJE-21-0901 -
Acta Crystallographica. Section D,... Nov 2021Thyroglobulin is a homodimeric glycoprotein that is essential for the generation of thyroid hormones in vertebrates. Upon secretion into the lumen of follicles in the...
Thyroglobulin is a homodimeric glycoprotein that is essential for the generation of thyroid hormones in vertebrates. Upon secretion into the lumen of follicles in the thyroid gland, tyrosine residues within the protein become iodinated to produce monoiodotyrosine (MIT) and diiodotyrosine (DIT). A subset of evolutionarily conserved pairs of DIT (and MIT) residues can then engage in oxidative coupling reactions that yield either thyroxine (T; produced from coupling of a DIT `acceptor' with a DIT `donor') or triiodothyronine (T; produced from coupling of a DIT acceptor with an MIT donor). Although multiple iodotyrosine residues have been identified as potential donors and acceptors, the specificity and structural context of the pairings (i.e. which donor is paired with which acceptor) have remained unclear. Here, single-particle cryogenic electron microscopy (cryoEM) was used to generate a high-resolution reconstruction of bovine thyroglobulin (2.3 Å resolution in the core region and 2.6 Å overall), allowing the structural characterization of two post-reaction acceptor-donor pairs as well as tyrosine residues modified as MIT and DIT. A substantial spatial separation between donor Tyr149 and acceptor Tyr24 was observed, suggesting that for thyroxine synthesis significant peptide motion is required for coupling at the evolutionarily conserved thyroglobulin amino-terminus.
Topics: Animals; Cattle; Cryoelectron Microscopy; Halogenation; Protein Conformation; Protein Domains; Protein Multimerization; Thyroglobulin
PubMed: 34726172
DOI: 10.1107/S2059798321010056 -
Acta Naturae 2021Early (preclinical) diagnosis of Parkinson's disease (PD) is a major challenge in modern neuroscience. The objective of this study was to experimentally evaluate a...
Early (preclinical) diagnosis of Parkinson's disease (PD) is a major challenge in modern neuroscience. The objective of this study was to experimentally evaluate a diagnostic challenge test with monoiodotyrosine (MIT), an endogenous inhibitor of tyrosine hydroxylase. Striatal dopamine was shown to decrease by 34% 2 h after subcutaneous injection of 100 mg/kg MIT to intact mice, with the effect not being amplified by a further increase in the MIT dose. The selected MIT dose caused motor impairment in a neurotoxic mouse model of preclinical PD, but not in the controls. This was because MIT reduced striatal dopamine to the threshold of motor symptoms manifestation only in PD mice. Therefore, using the experimental mouse model of preclinical PD, we have shown that a MIT challenge test may be used to detect latent nigrostriatal dysfunction.
PubMed: 34707902
DOI: 10.32607/actanaturae.11399 -
Biology Open Jun 2021Across the animal kingdom, dopamine plays a crucial role in conferring reinforcement signals that teach animals about the causal structure of the world. In the fruit fly...
Across the animal kingdom, dopamine plays a crucial role in conferring reinforcement signals that teach animals about the causal structure of the world. In the fruit fly Drosophila melanogaster, dopaminergic reinforcement has largely been studied using genetics, whereas pharmacological approaches have received less attention. Here, we apply the dopamine-synthesis inhibitor 3-Iodo-L-tyrosine (3IY), which causes acute systemic inhibition of dopamine signaling, and investigate its effects on Pavlovian conditioning. We find that 3IY feeding impairs sugar-reward learning in larvae while leaving task-relevant behavioral faculties intact, and that additional feeding of a precursor of dopamine (L-3,4-dihydroxyphenylalanine, L-DOPA), rescues this impairment. Concerning a different developmental stage and for the aversive valence domain. Moreover, we demonstrate that punishment learning by activating the dopaminergic neuron PPL1-γ1pedc in adult flies is also impaired by 3IY feeding, and can likewise be rescued by L-DOPA. Our findings exemplify the advantages of using a pharmacological approach in combination with the genetic techniques available in D. melanogaster to manipulate neuronal and behavioral function.
Topics: Animals; Behavior, Animal; Biosynthetic Pathways; Dopamine; Dopaminergic Neurons; Drosophila melanogaster; Larva; Learning; Monoiodotyrosine
PubMed: 34106227
DOI: 10.1242/bio.058198