An in silico study on human carcinogenicity mechanism of polybrominated biphenyls exposure.

Polybrominated biphenyls (PBBs) are associated with an increased risk of thyroid cancer; however, relevant mechanistic studies are lacking. In this study, we investigated the mechanisms underlying PBB-induced human thyroid cancer. Molecular docking and molecular dynamics methods were employed to investigate the metabolism of PBBs by the cytochrome P450 enzyme under aryl hydrocarbon receptor mediation into mono- and di-hydroxylated metabolites. This was taken as the molecular initiation event. Subsequently, considering the interactions of PBBs and their metabolites with the thyroxine-binding globulin protein as key events, an adverse outcome pathway for thyroid cancer caused by PBBs exposure was constructed. Based on 2D quantitative structure activity relationship (2D-QSAR) models, the contribution of amino acid residues and binding energy were analyzed to understand the mechanism underlying human carcinogenicity (adverse effect) of PBBs. Hydrogen bond and van der Waals interactions were identified as key factors influencing the carcinogenic adverse outcome pathway of PBBs. Analysis of non-bonding forces revealed that PBBs and their hydroxylation products were predominantly bound to the thyroxine-binding globulin protein through hydrophobic and hydrogen bond interactions. The key amino acids involved in hydrophobic interactions were alanine 330, arginine 381 and lysine 270, and the key amino acids involved in hydrogen bond interactions were arginine 381 and lysine 270. This study provides valuable insights into the mechanisms underlying human health risk associated with PBBs exposure.

Compound hemizygous variants in SERPINA7 gene cause thyroxine-binding globulin deficiency.

SUB-HEADING: Compound hemizygous variants in SERPINA7 gene. BACKGROUND: Thyroxine-binding globulin (TBG) is encoded by SERPINA7 (OMIM. 314200) which is located on Xq22.3. SERPINA7 variants caused TBG deficiency which does not require treatment, but the decreased thyroxine may be misdiagnosed as hypothyroidism. We discovered some variants of TBG caused by alterations that differ from previously reported. MATERIALS AND METHODS: In this study, we enrolled 32 subjects from 10 families and sequenced the SERPINA7 genes of TBG-deficient subjects. Then, variants were analyzed to assess their effect on TBG expression and secretion. Bioinformatics database, protein structure, and dynamics simulation were used to evaluate the deleterious effects. Finally, we identified 2 novel and 4 known variants, and found 26 of 30 subjects carried the p.L303F. The DynaMut predictions indicated the variants (p.E91K, p.I92T, p.R294C, and p.L303F) exhibited decreased stability. CONCLUSION: Analyses revealed the p.L303F change the protein stability and flexibility, and it had an impact on the function of TBG, but when coexisted with other variants it might change the conformational structure of the protein and aggravate the damage to the protein. We speculated that the existence of a higher number of variants resulted in lower TBG secretion.

Binding and activity of polybrominated diphenyl ether sulfates to thyroid hormone transport proteins and nuclear receptors.

Polybrominated diphenyl ethers (PBDEs) can be metabolized to hydroxylated PBDEs (OH-PBDEs), which play important roles in their disruption effects on the thyroid hormone (TH) system. Recently, multiple in vitro studies suggested that OH-PBDEs might be further metabolically transformed to PBDE sulfates. However, information about the bioactivity of PBDE sulfate metabolites is limited. In the present study, we explored the possible disruption effects of PBDE sulfates to the TH system by studying their binding and activity towards TH transport proteins and nuclear receptors. We found PBDE sulfates could bind to two major TH transport proteins (thyroxine-binding globulin and transthyretin). Besides, PBDE sulfates could also bind to two subtypes of TH nuclear receptors (TRα and TRß) and showed agonistic activity towards the subsequent signaling pathway. Moreover, the PBDE sulfates showed higher binding potency to TH transport proteins and TRs compared with their corresponding OH-PBDE precursors. Molecular docking results showed that replacement of hydroxyl groups with sulfate groups might lead to more hydrogen bond interactions with these proteins. Overall, our study suggested that PBDE sulfates might disturb the TH system by binding to TH transport proteins or TRs. Our finding indicated a possible mechanism for the TH system disruption effects of PBDEs through their sulfate metabolites.

Thyroxine-binding globulin deficiency due to a novel SERPINA7 mutation: Clinical characterization, analysis of X-chromosome inactivation pattern and protein structural modeling.

OBJECTIVE: Thyroxine-binding globulin (TBG) is the major human thyroid hormone transport protein, encoded by the SERPINA7 gene (Xq22.2). We aim to investigate the molecular basis of partial TBG deficiency (TBG-PD) in a female, by evaluating the X-chromosome inactivation pattern as well as the mutant protein structural modeling. DESIGN AND METHODS: Sequencing of the coding region of the SERPINA7 gene was performed in a female with a TBG-PD phenotype and her first-degree relatives. The proband presented with low serum levels of total T3 (TT3) and total T4 (TT4), serum TSH level of 5.4 µUI/mL (normal range, 0.35-5.5), and serum TBG level of 5.5 mg/L (normal range, 13.6-27.2). X-chromosome inactivation pattern was evaluated by methylation analysis of the androgen receptor gene (Xq11.2). Structural analysis of the SERPIN family was performed using Pymol and Areaimol, and PFSTATS for conservation analysis and family-wide investigation of equivalent positions in human homologs. Modeller was used for point mutation structural modeling. RESULTS: A novel missense SERPINA7 mutation (p.R35W; c.163C > T) was found in heterozygosity in the proband, and in hemizygosity in her affected siblings. The proband X-chromosome inactivation ratio was 20:80. The substitution of an arginine by a tryptophan is predicted to disrupt the protein surface and main electrostatic interactions. Tryptophans are extremely rare (0.1%) in this position. CONCLUSIONS: We report a new SERPINA7 variant associated with TBG-PD in three siblings. We named this variant TBG-Brasilia. The X-chromosome inactivation pattern may have accounted for the rare phenotypic expression in a female. The hydrophobic nature of the mutant is predicted to create an apolar patch at the surface, which results in protein aggregation and/or misfolding, potentially responsible for thyroid hormone transport defect.

Evolution of thyroid hormone distributor proteins.

Thyroid hormones (THs) are evolutionarily old hormones, having effects on metabolism in bacteria, invertebrates and vertebrates. THs bind specific distributor proteins (THDPs) to ensure their efficient distribution through the blood and cerebrospinal fluid in vertebrates. Albumin is a THDP in the blood of all studied species of vertebrates, so may be the original vertebrate THDP. However, albumin has weak affinity for THs. Transthyretin (TTR) has been identified in the blood across different lineages in adults vs juveniles. TTR has intermediate affinity for THs. Thyroxine-binding globulin has only been identified in mammals and has high affinity for THs. Of these THDPs, TTR is the only one known to be synthesised in the brain and is involved in moving THs from the blood into the cerebrospinal fluid. We analysed the rates of evolution of these three THDPs: TTR has been most highly conserved and albumin has had the highest rate of divergence.

Directional thyroid hormone distribution via the blood stream to target sites.

Thyroid hormones are bound to three major serum transport proteins, thyroxin-binding globulin (TBG), transthyretin (TTR) and human serum albumin (HSA). TBG has the strongest affinity for thyroid hormones, TTR is also found in the cerebrospinal fluid and HSA is the most abundant protein in plasma. Combination defects of either a high affinity TTR or HSA variant do not compensate TBG deficiency, underscoring the dominant role of TBG among the thyroid hormone transport proteins. On the other hand, coexistence of raised affinity TTR and HSA variants causes an augmented hyperthyroxinemia. Variations in thyroid hormone transport proteins may alter thyroid function tests to mimic hypo- or hyperthyroidism. As affected individuals are clinically euthyroid and do not require treatment, identification of thyroid hormone transport protein defects is important to avoid unnecessary diagnostic and therapeutic interventions. Mammals share the multilayered system of thyroid hormone binding proteins with humans. Some of them, especially carnivores, do not express TBG. In dogs, this defect has been shown to be caused by a defective hepatocyte nuclear factor-1 binding site in the TBG promoter, preventing TBG synthesis in the liver. The major endogenous thyroid hormone metabolite 3-iodothyronamine (3-T1AM) exerts marked cryogenic, metabolic, cardiac and central nervous system actions. It is bound to apolipoproteinB-100 (ApoB100), possibly facilitating its cellular uptake via interaction with the low density lipoprotein-receptor. This review summarizes the handling of hydrophobic charged thyroid hormone signaling molecules and their metabolite 3-T1AM in aqueous body fluids and the advantages and limits of their serum distributor proteins.

Chemistry and Biology in the Biosynthesis and Action of Thyroid Hormones.

Thyroid hormones (THs) are secreted by the thyroid gland. They control lipid, carbohydrate, and protein metabolism, heart rate, neural development, as well as cardiovascular, renal, and brain functions. The thyroid gland mainly produces l-thyroxine (T4) as a prohormone, and 5'-deiodination of T4 by iodothyronine deiodinases generates the nuclear receptor binding hormone T3. In this Review, we discuss the basic aspects of the chemistry and biology as well as recent advances in the biosynthesis of THs in the thyroid gland, plasma transport, and internalization of THs in their target organs, in addition to the deiodination and various other enzyme-mediated metabolic pathways of THs. We also discuss thyroid hormone receptors and their mechanism of action to regulate gene expression, as well as various thyroid-related disorders and the available treatments.

Surface plasmon resonance assay of inhibition by pharmaceuticals for thyroxine hormone binging to transport proteins.

We developed a surface plasmon resonance (SPR) assay to estimate the competitive inhibition by pharmaceuticals for thyroxine (T4) binding to thyroid hormone transport proteins, transthyretin (TTR) and thyroxine binding globulin (TBG). In this SPR assay, the competitive inhibition of pharmaceuticals for introducing T4 into immobilized TTR or TBG on the sensor chip can be estimated using a running buffer containing pharmaceuticals. The SPR assay showed reproducible immobilization of TTR and TBG, and the kinetic binding parameters of T4 to TTR or TBG were estimated. The equilibrium dissociation constants of TTR or TBG measured by SPR did not clearly differ from data reported for other binding assays. To estimate the competitive inhibition of tetraiodothyroacetic acid, diclofenac, genistein, ibuprofen, carbamazepine, and furosemide, reported to be competitive or noncompetitive pharmaceuticals for T4 binding to TTR or TBG, their 50% inhibition concentrations (IC50) (or 80% inhibition concentration, IC80) were calculated from the change of T4 responses in sensorgrams obtained with various concentrations of the pharmaceuticals. Our SPR method should be a useful tool for predicting the potential of thyroid toxicity of pharmaceuticals by evaluating the competitive inhibition of T4 binding to thyroid hormone binding proteins, TTR and TBG.

Novel mutation p.A64D in the Serpina7 gene as a cause of partial thyroxine-binding globulin deficiency associated with increases affinity in transthyretin by a known p.A109T mutation in the TTR gene.

Partial thyroxine-binding globulin deficiency (TBG-PD) is an endocrine defect with a prevalence of 1:4 000 in newborns. Due to the presence of a single TBG gene on the X chromosome, most familial TBG defects follow an X-linked inheritance pattern. Abnormal T4 binding to T4-binding prealbumin (TTR) is a rare cause of euthyroid hyperthyroxinemia, which is transmitted by autosomal dominant inheritance. The purpose of the present study was to identify and characterize new mutations in the Serpina7 and TTR genes in a complete family with typical TBG-PD. All patients underwent clinical and biochemical evaluation. Sequencing of DNA, population screening by (SSCP) analysis, and bioinformatics studies were performed. Molecular studies revealed a novel p.A64D mutation in the exon 1 of Serpina7 gene associated with the previously reported p.A109T mutation in the exon 4 of TTR gene. To our knowledge, this is the first report of a patient with a TBG-PD by a mutation in Serpina7 that was coincident with a mutation in TTR gene that increased affinity of TTR for T4. This work contributes to elucidate the molecular basis of the defects of thyroid hormone transport in serum and the improvement of the diagnosis avoiding unnecessary therapy.

[Thyroid hormones and their precursors I. Biochemical properties]. / Pajzsmirigyhormonok és eloanyagaik I. Biokémiai tulajdonságok.

This paper and the following one (see the next issue of Acta Pharmaceutica Hungarica) survey the biological roles and the related site-specific physico-chemical parameters (basicity and lipophilicity) of the presently known thyroid hormones (thyroxine, liothyronine and reverse liothyronine) and their biological precursors (monoiodotyrosine and diiodotyrosine). Here the literature of the thyroid hormone biochemistry, biosynthesis, plasma- and membrane transport is summarized, focusing on the pH-dependent processes. Biosyntheses of the thyroid hormones take place by oxidative coupling of two iodotyrosine residues catalyzed by thyreoperoxidase in thyreoglobulin. The protonation state of the precursors, especially that of the phenolic OH is crucial for the biosynthesis, since anionic iodotyrosine residues can only be coupled in the thyroid hormone biosyntheses. In the blood more than 99% of the circulating thyroid hormone is bound to plasma proteins among which the thyroxine-binding globulin and transthyretin are crucial. The amphiphilic character of the hormones is assumed to be the reason why their membrane transport is an energy-dependent, transport-mediated process, in which the organic anion transporter family, mainly OATP1C1, and the amino acid transporters, such as MCT8 play important roles. Liothyronine is the biologically active hormone; it binds the thyroid hormone receptor, a type of nuclear receptor. There are two major thyroid hormone receptor (TR) isoforms, alfa (TRalpha) and beta (TRbeta). The activation of the TRalpha is associated with modifications in cardiac behavior, while activation of the TRbeta is associated with increasing metabolic rates, resulting in weight loss and reduction of blood plasma lipid levels. The affinity of the thyroid hormones for different proteins depends on the ionization state of the ligands. The site-specific physico-chemical characterization of the thyroid hormones is of fundamental importance to understand their (patho)physiological behavior and also, to influence their therapeutic properties at the molecular level.

The allosteric modulation of thyroxine-binding globulin affinity is entropy driven.

BACKGROUND: Thyroxine-binding globulin (TBG) is a non-inhibitory member of the serpin family of proteins whose main structural element is the reactive center loop (RCL), that, upon cleavage by proteases, is inserted into the protein core adopting a ß-strand conformation (stressed to relaxed transition, S-to-R). After S-to-R transition thyroxine (T4) affinity decreases. However, crystallographic studies in the presence or absence of the hormone in different states are unable to show significant differences in the structure and interactions of the binding site. Experimental results also suggest the existence of several S states (differing in the number of inserted RCL residues), associated with a differential affinity. METHODS: To shed light into the molecular basis that regulates T4 affinity according to the degree of RCL insertion in TBG, we performed extended molecular dynamics simulations combined with several thermodynamic analysis of the T4 binding to TBG in three different S states, and in the R state. RESULTS: Our results show that, despite T4 binding in the protein by similar interactions in all states, a good correlation between the degree of RCL insertion and the binding affinity, driven by a change in TBG conformational entropy, was observed. CONCLUSION: TBG allosteric regulation is entropy driven. The presence of multiple S states may allow more efficient T4 release due to protease activity. GENERAL SIGNIFICANCE: The presented results are clear examples of how computer simulation methods can reveal the thermodynamic basis of allosteric effects, and provide a general framework for understanding serpin allosteric affinity regulation.

Serpins as hormone carriers: modulation of release.

The hormone-carrying serpins, thyroxine- and corticosteroid-binding globulins, TBG and CBG, provide a clear example of the way the serpin conformational mechanism can be adapted not only to give an irreversible switching-off of function but also more significantly to allow a constant dynamic modulation of activity. This is illustrated here with the demonstration that hormone release from both TBG and CBG is responsive to changes in ambient temperature and specifically to changes in body temperature. An exception to this adaptation of the serpin mechanism is seen with another family member, angiotensinogen, in which hormone release is modulated by a redox switch and is apparently independent of changes in the serpin framework.

Allosteric modulation of hormone release from thyroxine and corticosteroid-binding globulins.

The release of hormones from thyroxine-binding globulin (TBG) and corticosteroid-binding globulin (CBG) is regulated by movement of the reactive center loop in and out of the ß-sheet A of the molecule. To investigate how these changes are transmitted to the hormone-binding site, we developed a sensitive assay using a synthesized thyroxine fluorophore and solved the crystal structures of reactive loop cleaved TBG together with its complexes with thyroxine, the thyroxine fluorophores, furosemide, and mefenamic acid. Cleavage of the reactive loop results in its complete insertion into the ß-sheet A and a substantial but incomplete decrease in binding affinity in both TBG and CBG. We show here that the direct interaction between residue Thr(342) of the reactive loop and Tyr(241) of the hormone binding site contributes to thyroxine binding and release following reactive loop insertion. However, a much larger effect occurs allosterically due to stretching of the connecting loop to the top of the D helix (hD), as confirmed in TBG with shortening of the loop by three residues, making it insensitive to the S-to-R transition. The transmission of the changes in the hD loop to the binding pocket is seen to involve coherent movements in the s2/3B loop linked to the hD loop by Lys(243), which is, in turn, linked to the s4/5B loop, flanking the thyroxine-binding site, by Arg(378). Overall, the coordinated movements of the reactive loop, hD, and the hormone binding site allow the allosteric regulation of hormone release, as with the modulation demonstrated here in response to changes in temperature.

Thyroid function tests during first-trimester of pregnancy: a review of literature.

This literature review was conducted to summarize the main points of maternal thyroid function tests, with particular attention in the first trimester of pregnancy which accompanied with significant biochemical and metabolic alteration. The evaluation of thyroid function of either hyperthyroidism and hypothyroidism should be assessed by determination of serum Thyroid Stimulating Hormone (TSH), Thyroxine (T4), Triiodothyronine (T3), Iodine and Thyroid Autoantibodies. Glomerular filtration rate is increased during pregnancy; therefor iodine deficiency should be evaluated during the pregnancy to prevent hypothyroidism. The role which can be played by Human Chronic Gonadotropin (hCG) on stimulating the thyroid gland to become over-active was investigated. Serum level ofthyroglobulin (Tg) and Thyroxin Binding Globulin (TBG) should be assessed for proper assessments of thyroid gland during pregnancy. Thyroid function tests during first-trimester of pregnancy and particularly the reference interval for thyroid function tests for pregnant women in each region has to be established, to prevent mis-diagnosis and irreversible mental and physical adverse affect for growing fetus.

Molecular and structural basis of steroid hormone binding and release from corticosteroid-binding globulin.

Corticosteroid-binding globulin (CBG), a non-inhibitory member of the serine proteinase inhibitor (serpin) super-family, is the high-affinity transport protein for glucocorticoids in vertebrate blood. Plasma CBG is a glycoprotein with 30% of its mass represented by N-linked oligosaccharide chains. Its well-characterized steroid-binding properties represent a "bench-mark data set" used extensively for in silico studies of protein-ligand interactions and drug design. Recent crystal structure analyses of intact rat CBG and cleaved human CBG have revealed the precise topography of the steroid-binding site, and shown that cortisol-bound CBG displays a typical stressed (S) serpin conformation with the reactive center loop (RCL) fully exposed from the central beta-sheet A, while proteolytic cleavage of the RCL results in CBG adopting a relaxed (R) conformation with the cleaved RCL fully inserted within the protein core. These crystal structures have set the stage for mechanistic studies of CBG function which have so far shown that helix D plays a key role in coupling RCL movement and steroid-binding site integrity, and provided evidence for an allosteric mechanism that modulates steroid binding and release from CBG. These studies have also revealed how the irreversible release of steroids occurs after proteolysis and re-orientation of the RCL within the R conformation. This recent insight into the structure and function of CBG reveals how naturally occurring genetic CBG mutations affect steroid binding, and helps understand how proteolysis of CBG enhances the targeted delivery of biologically active steroids to their sites of action.