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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 -
The Journal of Biological Chemistry Jul 1990Lysosomal transport of monoiodotyrosine was characterized in countertransport experiments using rat FRTL-5 thyroid cell lysosomes. Monoiodotyrosine carrier activity was...
Lysosomal transport of monoiodotyrosine was characterized in countertransport experiments using rat FRTL-5 thyroid cell lysosomes. Monoiodotyrosine carrier activity was temperature-dependent (Ea = 11.65 kcal/mol) and had a pH optimum of 7.5. Carrier activity was minimally inhibited by KCl and NaCl, but unaffected by the presence of other ions or ATP. Monoiodotyrosine transport was unaffected by the presence of carbonyl cyanide m-chlorophenylhydrazone, nigericin, or ammonium chloride, indicating that a proton or K+ gradient is not necessary for monoiodotyrosine transport across the lysosomal membrane. Monoiodotyrosine countertransport showed a 6-fold increase in lysosomes from FRTL-5 cells grown in medium containing thyrotropin by comparison to cells grown without this hormone. Thyrotropin responsiveness raised the possibility that monoiodotyrosine was transported by system h, the only known lysosomal carrier whose activity is enhanced by thyrotropin. Consistent with this, monoiodotyrosine-loaded lysosomes exhibited countertransport of [3H]tyrosine, [3H]phenylalanine, and [3H]leucine, three system h ligands, but not [3H]cystine, a nonsystem h ligand. Unlabeled tyrosine, phenylalanine, and leucine, but not cystine or proline, inhibited [125I]monoiodotyrosine countertransport, and leucine inhibition of [3H]tyrosine countertransport and [125I]monoiodotyrosine countertransport yielded virtually identical KI values, 3.5 and 3.2 microM, respectively. Competition studies with monoiodotyrosine analogues showed that system h recognizes a broad range of ligands with an alpha-amino acid configuration at one end and a hydrophobic region at the other. Ring-substituted halogens, regardless of mass or ring position, but not amino, nitro, hydroxy, or methoxy groups, enhanced carrier recognition of system h analogues. It appears that a single system effects the transport of iodinated (e.g. monoiodotyrosine) and noniodinated (e.g. tyrosine) thyroglobulin catabolites into the cytosol for salvage and reutilization by FRTL-5 thyroid cells.
Topics: Animals; Binding, Competitive; Biological Transport; Carrier Proteins; Cell Line; Hot Temperature; Hydrogen-Ion Concentration; Kinetics; Leucine; Lysosomes; Monoiodotyrosine; Phenylalanine; Rats; Structure-Activity Relationship; Thyroid Gland; Thyrotropin; Tyrosine
PubMed: 2358448
DOI: No ID Found -
California Medicine Jan 1959Five to 10 per cent of cretinism in the United States is due to some congenital enzymatic defect in thyroid hormone synthesis. The clinical signs of hypothyroidism...
Five to 10 per cent of cretinism in the United States is due to some congenital enzymatic defect in thyroid hormone synthesis. The clinical signs of hypothyroidism appear in early infancy. Differentiation from athyreotic cretinism is important because the metabolic defect tends to be familial and its presence in the patient's infant relatives should be diagnosed as early as possible. The differentiation is easily made if a goiter is discernible, but if it is not, radioiodine uptake should be measured, for in this condition the uptake is normal or greater. Thyroid replacement is the treatment in either the athyreotic state or the metabolic deficiency. The three known defects in thyroid hormone synthesis are (1) failure to oxidize iodine to elemental iodine resulting in failure of all subsequent processes; (2) failure to deiodinate free iodotyrosine, and (3) failure to form iodothyronine although the previous steps are accomplished.
Topics: Congenital Hypothyroidism; Goiter; Humans; Hypothyroidism; Infant; Iodides; Iodine; Iodine Radioisotopes; Monoiodotyrosine; Syndrome; Thyroid Hormones
PubMed: 13618742
DOI: No ID Found -
Frontiers in Bioscience (Landmark... Jan 2019A phylogenetically conserved 5-residue thyroid hormone (TH)- binding motif was originally found in a few TH plasma carriers and, more recently, in all known plasma and...
A phylogenetically conserved 5-residue thyroid hormone (TH)- binding motif was originally found in a few TH plasma carriers and, more recently, in all known plasma and cell-associated proteins interacting with TH as well as in proteins involved in iodide uptake. Minor variations of the motif were found, depending on the particular class of those proteins. Since thyroglobulin (Tg) is the protein matrix for TH synthesis starting from iodination of a selected number of tyrosines (to form first monoiodotyrosine (MIT) and diiodotyrosine (DIT) and then T3 and T4), we hypothesized that by searching the presence of perfect or imperfect versions of that motif in two Tg species (human and murine) in which the iodinated tyrosines and pattern of iodotyrosine/iodothyronine formation are known, we could have found relevant explanations. Explanations, which are not furnished by the simple possession of tyrosine-iodination motifs and sequence of the iodination motif, concern why only some (but not other) tyrosine residues in one species are iodinated and why they have a particular iodination pattern. In this bioinformatics study, we provide such explanations.
Topics: Amino Acid Motifs; Amino Acid Sequence; Animals; Binding Sites; Computational Biology; Diiodotyrosine; Humans; Iodine; Mice; Monoiodotyrosine; Protein Binding; Thyroglobulin; Thyroid Hormones; Thyronines
PubMed: 30468652
DOI: 10.2741/4714 -
The Journal of Biological Chemistry Nov 1982Stopped flow experiments were carried out with purified hog thyroid peroxidase (A413 nm/A280 nm = 0.42). In the steady state of oxidations of L- and D-tyrosines,...
Stopped flow experiments were carried out with purified hog thyroid peroxidase (A413 nm/A280 nm = 0.42). In the steady state of oxidations of L- and D-tyrosines, N-acetyltyrosinamide, and monoiodotyrosine, thyroid peroxidase existed in the form of Compound I, the primary catalytic intermediate of peroxidase in its reaction with H2O2. Kinetic results led us to conclude that thyroid peroxidase catalyzes two-electron oxidations of these molecules. In the steady state of oxidation of diiodotyrosine, on the other hand, the enzyme was found in the form of compound II at pH 7.4, but in the form of compound I at pH 5.5. The result implies that the mechanism of diiodotyrosine oxidation varied from a one-electron to a two-electron type as the pH decreased. The selection of mechanisms of oxidation appears to be peculiar to thyroid peroxidase; horseradish peroxidase and lactoperoxidase catalyzed only one-electron oxidations of these five donor molecules. Rate constants for rate-limiting steps in the reactions of these donor molecules with the three peroxidases were measured by overall kinetic and stopped flow kinetic methods.
Topics: Animals; Diiodotyrosine; Electron Transport; Horseradish Peroxidase; Iodide Peroxidase; Kinetics; Lactoperoxidase; Monoiodotyrosine; Oxidation-Reduction; Peroxidases; Substrate Specificity; Swine; Thyroid Gland; Tyrosine
PubMed: 7142155
DOI: No ID Found -
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 -
The Journal of Biological Chemistry Jun 1975Insulin was enzymatically moniodinated with 127-I or 125-I, and an improved method of purification by anion exchange chromatography was employed. (127-I)Monoiodoinsulin...
Insulin was enzymatically moniodinated with 127-I or 125-I, and an improved method of purification by anion exchange chromatography was employed. (127-I)Monoiodoinsulin was identified by spectrophotometric analysis and its molar extinction coefficient determined to be 6.31 times 10-3 M-1 cm minus 1. The observed specific activity of carrier-free (125-I)monoidoinsulin was close to the theoretical value (378mCi/mg). The monoiodotyrosyl residue of monoidoinsulin was shown to be solvent-exposed. The ionic properties of the substituted hormone were altered at pH values close to the pK of monoiodotyrosine (8.85), but the pI was unchanged (5.65). (127-I)Monoiodoinsulin formed rhombohedral crystals and co-crystallized with native insulin. Monoidoinsulin was indistinguishable from insulin with respect to binding to two out of three guinea pig anti-insulin sera, to binding to IM9 cultured human lymphocytes, and to binding to isolated rat hepatocyte plasma membranes. The potency of monoidoinsulin was not statistically different from that of insulin in the rat fat cell bioassay and in the mouse convulsion assay. An insulin-degrading enzyme extracted from rat liver degraded monoiodoinsulin less readily than native insulin; monoiodoinsulin was a competitive inhibitor of insulin degradation, and the Km values were 30 nM AND 78 NM for monoidoinsulin and native insulin, respectively. It is concluded that monoidination does not markedly alter the three-dimensional structure of the molecule and that only a few sensitive biological systems are able to distinguish the monoidinated from the native hormone.
Topics: Adipose Tissue; Animals; Biological Assay; Cattle; Cell Membrane; Chromatography, Ion Exchange; Crystallization; Electrophoresis, Polyacrylamide Gel; Guinea Pigs; Hydrogen-Ion Concentration; Immunoassay; Insulin; Insulin Antibodies; Insulysin; Iodine; Iodine Isotopes; Iodine Radioisotopes; Isoelectric Focusing; Liver; Lymphocytes; Methods; Peptide Hydrolases; Radioimmunoassay; Receptors, Cell Surface; Urea
PubMed: 236316
DOI: No ID Found -
The Biochemical Journal 1944
PubMed: 16747803
DOI: 10.1042/bj0380320 -
Hematology, Transfusion and Cell Therapy 2021Although the efficacy of hydroxyurea (HU) in inhibiting erythrocyte sickling has been well demonstrated, the action of this drug on human neutrophils and the mechanism...
INTRODUCTION
Although the efficacy of hydroxyurea (HU) in inhibiting erythrocyte sickling has been well demonstrated, the action of this drug on human neutrophils and the mechanism by which it improves the manifestations of the disease have not been studied thoroughly. We aimed to investigate the cell viability, along with inflammatory and oxidative markers in the neutrophils of sickle cell anemia (SCA) patients and the effects of HU therapy on these cells, by evaluating the dose-responsiveness.
METHODS
In the present study, 101 patients (45 men and 56 women, aged 18-69 years) with SCA were divided into groups according to the use or not of HU: the SS group (without HU treatment, n = 47) and the SSHU group (under HU treatment, n = 54). The SSHU group was further stratified into subgroups according to the daily dose of the drug that patients already used: SSHU - 0.5 g (n = 19); SSHU - 1 g (n = 26) and SSHU - 1.5-2 g (n = 9). A control group (AA) comprised 50 healthy individuals. Neutrophils isolated from whole blood were analyzed using Trypan Blue, monoiodotyrosine (MTT) and lactate dehydrogenase (LDH) toxicity assays. Myeloperoxidase (MPO), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) activities and concentrations of interleukin 10 (IL-10), tumor necrosis factor alpha (TNF-α) and malonaldehyde (MDA) were also measured.
RESULTS
Neutrophils from SCA patients showed membrane fragility and a significant decrease in cell viability when analyzed by Trypan Blue (p < 0.05), MTT (p < 0.001) and LDH (p = 0.011), compared to the AA group. Levels of inflammatory (MPO, TNF-α, and IL-10) and oxidative markers (SOD, GSH-Px, and MDA) were also altered (p < 0.05) in these cells, showing a significant difference in the SSHU-1g and SSHU - 1.5-2 g groups, compared to the SS group. Treatment with HU reverted the levels of all markers to concentrations similar to those in healthy individuals in a positive dose-effect relationship.
CONCLUSION
The HU did not generate a cytotoxic effect on neutrophils in SCA patients, but it modulated their oxidative and inflammatory mechanisms, promoting cytoprotection with a positive dose-effect.
PubMed: 33051133
DOI: 10.1016/j.htct.2020.07.011