Iodine Atoms: A New Molecular Feature for the Design of Potent Transthyretin Fibrillogenesis Inhibitors

The thyroid hormone and retinol transporter protein known as transthyretin (TTR) is in the origin of one of the 20 or so known amyloid diseases. TTR self assembles as a homotetramer leaving a central hydrophobic channel with two symmetrical binding sites. The aggregation pathway of TTR into amiloid fibrils is not yet well characterized but in vitro binding of thyroid hormones and other small organic molecules to TTR binding channel results in tetramer stabilization which prevents amyloid formation in an extent which is proportional to the binding constant. Up to now, TTR aggregation inhibitors have been designed looking at various structural features of this binding channel others than its ability to host iodine atoms. In the present work, greatly improved inhibitors have been designed and tested by taking into account that thyroid hormones are unique in human biochemistry owing to the presence of multiple iodine atoms in their molecules which are probed to interact with specific halogen binding domains sitting at the TTR binding channel. The new TTR fibrillogenesis inhibitors are based on the diflunisal core structure because diflunisal is a registered salicylate drug with NSAID activity now undergoing clinical trials for TTR amyloid diseases. Biochemical and biophysical evidence confirms that iodine atoms can be an important design feature in the search for candidate drugs for TTR related amyloidosis.     

Novel Transthyretin Amyloid Fibril Formation Inhibitors: Synthesis,Biological Evaluation,and X-Ray Structural Analysis

Transthyretin (TTR) is one of thirty non-homologous proteins whose misfolding, dissociation, aggregation, and deposition is linked to human amyloid diseases. Previous studies have identified that TTR amyloidogenesis can be inhibited through stabilization of the native tetramer state by small molecule binding to the thyroid hormone sites of TTR. We have evaluated a new series of β-aminoxypropionic acids (compounds 5–21), with a single aromatic moiety (aryl or fluorenyl) linked through a flexible oxime tether to a carboxylic acid. These compounds are structurally distinct from the native ligand thyroxine and typical halogenated biaryl NSAID-like inhibitors to avoid off-target hormonal or anti-inflammatory activity. Based on an in vitro fibril formation assay, five of these compounds showed significant inhibition of TTR amyloidogenesis, with two fluorenyl compounds displaying inhibitor efficacy comparable to the well-known TTR inhibitor diflunisal. Fluorenyl 15 is the most potent compound in this series and importantly does not show off-target anti-inflammatory activity. Crystal structures of the TTR∶inhibitor complexes, in agreement with molecular docking studies, revealed that the aromatic moiety, linked to the sp²-hybridized oxime carbon, specifically directed the ligand in either a forward or reverse binding mode. Compared to the aryl family members, the bulkier fluorenyl analogs achieved more extensive interactions with the binding pockets of TTR and demonstrated better inhibitory activity in the fibril formation assay. Preliminary optimization efforts are described that focused on replacement of the C-terminal acid in both the aryl and fluorenyl series (compounds 22–32). The compounds presented here constitute a new class of TTR inhibitors that may hold promise in treating amyloid diseases associated with TTR misfolding.     

The effect of iodide and chloride on transthyretin structure and stability

Transthyretin amyloid formation occurs through a process of tetramer destabilization and partial unfolding. Small molecules, including the natural ligand thyroxine, stabilize the tetrameric form of the protein, and serve as inhibitors of amyloid formation. Crucial for TTR's ligand-binding properties are its three halogen-binding sites situated at the hormone-binding channel. In this study, we have performed a structural characterization of the binding of two halides, iodide and chloride, to TTR. Chlorides are known to shield charge repulsions at the tetrameric interface of TTR, which improve tetramer stability of the protein. Our study shows that iodides, like chlorides, provide tetramer stabilization in a concentration-dependent manner and at concentrations approximately 15-fold below that of chlorides. To elucidate binding sites of the halides, we took advantage of the anomalous scattering of iodide and used the single-wavelength anomalous dispersion (SAD) method to solve the iodide-bound TTR structure at 1.8 A resolution. The structure of chloride-bound TTR was determined at 1.9 A resolution using difference Fourier techniques. The refined structures showed iodides and chlorides bound at two of the three halogen-binding sites located at the hydrophobic channel. These sites therefore also function as halide-binding sites.     

Crystallographic Study of Novel Transthyretin Ligands Exhibiting Negative-Cooperativity between Two Thyroxine Binding Sites

Background

Transthyretin (TTR) is a homotetrameric serum and cerebrospinal fluid protein that transports thyroxine (T4) and retinol by binding to retinol binding protein. Rate-limiting tetramer dissociation and rapid monomer misfolding and disassembly of TTR lead to amyloid fibril formation in different tissues causing various amyloid diseases. Based on the current understanding of the pathogenesis of TTR amyloidosis, it is considered that the inhibition of amyloid fibril formation by stabilization of TTR in native tetrameric form is a viable approach for the treatment of TTR amyloidosis.

Methodology and Principal Findings

We have examined interactions of the wtTTR with a series of compounds containing various substitutions at biphenyl ether skeleton and a novel compound, previously evaluated for binding and inhibiting tetramer dissociation, by x-ray crystallographic approach. High resolution crystal structures of five ligands in complex with wtTTR provided snapshots of negatively cooperative binding of ligands in two T4 binding sites besides characterizing their binding orientations, conformations, and interactions with binding site residues. In all complexes, the ligand has better fit and more potent interactions in first T4 site i.e. (AC site) than the second T4 site (BD site). Together, these results suggest that AC site is a preferred ligand binding site and retention of ordered water molecules between the dimer interfaces further stabilizes the tetramer by bridging a hydrogen bond interaction between Ser117 and its symmetric copy.

Conclusion

Novel biphenyl ether based compounds exhibit negative-cooperativity while binding to two T4 sites which suggests that binding of only single ligand molecule is sufficient to inhibit the TTR tetramer dissociation.     

The binding of synthetic triiodo l-thyronine analogs to human transthyretin: molecular basis of cooperative and non-cooperative ligand recognition

Transthyretin (TTR) is a tetrameric β-sheet-rich transporter protein directly involved in human amyloid diseases. Several classes of small molecules can bind to TTR delaying its amyloid fibril formation, thus being promising drug candidates to treat TTR amyloidoses. In the present study, we characterized the interactions of the synthetic triiodo L-thyronine analogs and thyroid hormone nuclear receptor TRβ-selective agonists GC-1 and GC-24 with the wild type and V30M variant of human transthyretin (TTR). To achieve this aim, we conducted in vitro TTR acid-mediated aggregation and isothermal titration calorimetry experiments and determined the TTR:GC-1 and TTR:GC-24 crystal structures. Our data indicate that both GC-1 and GC-24 bind to TTR in a non-cooperative manner and are good inhibitors of TTR aggregation, with dissociation constants for both hormone binding sites (HBS) in the low micromolar range. Analysis of the crystal structures of TTRwt:GC-1(24) complexes and their comparison with the TTRwt X-ray structure bound to its natural ligand thyroxine (T4) suggests, at the molecular level, the basis for the cooperative process displayed by T4 and the non-cooperative process provoked by both GC-1 and GC-24 during binding to TTR.     

Iodination of salicylic acid improves its binding to transthyretin

Transthyretin (TTR) is a plasma homotetrameric protein associated with senile systemic amyloidosis and familial amyloidotic polyneuropathy. In theses cases, TTR dissociation and misfolding induces the formation of amyloidogenic intermediates that assemble into toxic oligomeric species and lead to the formation of fibrils present in amyloid deposits. The four TTR monomers associate around a central hydrophobic channel where two thyroxine molecules can bind simultaneously. In each thyroxine binding site there are three pairs of symmetry related halogen binding pockets which can accommodate the four iodine substituents of thyroxine. A number of structurally diverse small molecules that bind to the TTR channel increasing the protein stability and thereafter inhibiting amyloid fibrillogenesis have been tested. In order to take advantage of the high propensity to interactions between iodine substituents and the TTR channel we have identified two iodinated derivatives of salicylic acid, 5-iodosalicylic acid and 3,5-diiodosalicylic acid, available commercially. We report in this paper the relative binding affinities of salicylic acid and the two iodinated derivatives and the crystal structure of TTR complexed with 3,5-diiodosalicylic acid, to elucidate the higher binding affinity of this compound towards TTR.     

Inhibition of transthyretin amyloid fibril formation by 2,4-dinitrophenol through tetramer stabilization

Transthyretin (TTR), a homotetrameric thyroxine transport protein found in the plasma and cerebrospinal fluid, circulates normally as a innocuous soluble protein. In some individuals, TTR polymerizes to form insoluble amyloid fibrils. TTR amyloid fibril formation and deposition have been associated with several diseases like familial amyloid polyneuropathy and senile systemic amyloidosis. Inhibition of the fibril formation is considered a potential strategy for the therapeutic intervention. The effect of small water-soluble, hydrophobic ligand 2,4-dinitrophenol (2,4-DNP) on TTR amyloid formation has been tested. 2,4-DNP binds to TTR both at acidic and physiological pH, as shown by the quenching of TTR intrinsic fluorescence. Interestingly, 2,4-DNP not only binds to TTR at acidic pH but also inhibits amyloid fibril formation as shown by the light scattering and Congo red-binding assay. Inhibition of fibril formation by 2,4-DNP appears to be through the stabilization of TTR tetramer upon binding to the protein, which includes active site. These findings may have implications for the development of mechanism based small molecular weight compounds as therapeutic agents for the prevention/inhibition of the amyloid diseases.     

The Flavonoid Luteolin,but Not Luteolin-7-O-Glucoside,Prevents a Transthyretin Mediated Toxic Response

Transthyretin (TTR) is a homotetrameric plasma protein with amyloidogenic properties that has been linked to the development of familial amyloidotic polyneuropathy (FAP), familial amyloidotic cardiomyopathy, and senile systemic amyloidosis. The in vivo role of TTR is associated with transport of thyroxine hormone T4 and retinol-binding protein. Loss of the tetrameric integrity of TTR is a rate-limiting step in the process of TTR amyloid formation, and ligands with the ability to bind within the thyroxin binding site (TBS) can stabilize the tetramer, a feature that is currently used as a therapeutic approach for FAP. Several different flavonoids have recently been identified that impair amyloid formation. The flavonoid luteolin shows therapeutic potential with low incidence of unwanted side effects. In this work, we show that luteolin effectively attenuates the cytotoxic response to TTR in cultured neuronal cells and rescues the phenotype of a Drosophila melanogaster model of FAP. The plant-derived luteolin analogue cynaroside has a glucoside group in position 7 of the flavone A-ring and as opposed to luteolin is unable to stabilize TTR tetramers and thus prevents a cytotoxic effect. We generated high-resolution crystal-structures of both TTR wild type and the amyloidogenic mutant V30M in complex with luteolin. The results show that the A-ring of luteolin, in contrast to what was previously suggested, is buried within the TBS, consequently explaining the lack of activity from cynaroside. The flavonoids represent an interesting group of drug candidates for TTR amyloidosis. The present investigation shows the potential of luteolin as a stabilizer of TTR in vivo. We also show an alternative orientation of luteolin within the TBS which could represent a general mode of binding of flavonoids to TTR and is of importance concerning the future design of tetramer stabilizing drugs.     

Post‐translational modifications of transthyretin affect the triiodonine‐binding potential

Transthyretin (TTR) is a visceral protein, which facilitates the transport of thyroid hormones in blood and cerebrospinal fluid. The homotetrameric structure of TTR enables the simultaneous binding of two thyroid hormones per molecule. Each TTR subunit provides a single cysteine residue (Cys₁₀), which is frequently affected by oxidative post‐translational modifications. As Cys₁₀ is part of the thyroid hormone‐binding channel within the TTR molecule, PTM of Cys₁₀ may influence the binding of thyroid hormones. Therefore, we analysed the effects of Cys₁₀ modification with sulphonic acid, cysteine, cysteinylglycine and glutathione on binding of triiodothyronine (T3) by molecular modelling. Furthermore, we determined the PTM pattern of TTR in serum of patients with thyroid disease by immunoprecipitation and mass spectrometry to evaluate this association in vivo. The in silico assays demonstrated that oxidative PTM of TTR resulted in substantial reorganization of the intramolecular interactions and also affected the binding of T3 in a chemotype‐ and site‐specific manner with S‐glutathionylation as the most potent modulator of T3 binding. These findings were supported by the in vivo results, which indicated thyroid function‐specific patterns of TTR with a substantial decrease in S‐sulphonated, S‐cysteinylglycinated and S‐glutathionylated TTR in hypothyroid patients. In conclusion, this study provides evidence that oxidative modifications of Cys₁₀ seem to affect binding of T3 to TTR probably because of the introduction of a sterical hindrance and induction of conformational changes. As oxidative modifications can be dynamically regulated, this may represent a sensitive mechanism to adjust thyroid hormone availability.     

Transthyretin chemical chaperoning by flavonoids: Structure–activity insights towards the design of potent amyloidosis inhibitors

BackgroundMany polyphenols have been proposed as broad-spectrum inhibitors of amyloid formation. To investigate structure–activity relationships relevant for the interaction of flavonoids with transthyretin (TTR), the protein associated with familial amyloid polyneuropathy (FAP), we compared the effects of major tea catechins and their larger polymers theaflavins, side-by-side, on TTR amyloid formation process.MethodsInteraction of flavonoids with TTR and effect on TTR stability were assessed through binding assays and isoelectric focusing in polyacrylamide gel. TTR aggregation was studied, in vitro, by dynamic light scattering (DLS), transmission electron microscopy (TEM) and in cell culture, through cytotoxicity assays.ResultsTested flavonoids bound to TTR and stabilized the TTR tetramer, with different potencies. The flavonoids also inhibited in vitro formation of TTR small oligomeric species and in cell culture inhibited pathways involving caspase-3 activation and ER stress that are induced by TTR oligomers. In all assays performed the galloyl esters presented higher potency to inhibit aggregation than the non-gallated flavonoids tested.ConclusionsOur results highlight the presence of gallate ester moiety as key structural feature of flavonoids in chemical chaperoning of TTR aggregation. Upon binding to the native tetramer, gallated flavonoids redirect the TTR amyloidogenic pathway into unstructured nontoxic aggregation assemblies more efficiently than their non-gallated forms.General significanceOur findings suggest that galloyl moieties greatly enhance flavonoid anti-amyloid chaperone activity and this should be taken into consideration in therapeutic candidate drug discovery.     

Change in structure of the N-terminal region of transthyretin produces change in affinity of transthyretin to T4 and T3

The relationship between the structure of the N-terminal sequence of transthyretin (TTR) and the binding of thyroid hormone was studied. A recombinant human TTR and two derivatives of Crocodylus porosus TTRs, one with the N-terminal sequence replaced by that of human TTR (human/crocTTR), the other with the N-terminal segment removed (truncated crocTTR), were synthesized in Pichia pastoris. Subunit mass, native molecular weight, tetramer formation, cross-reactivity to TTR antibodies and binding to retinol-binding protein of these recombinant TTRs were similar to TTRs found in nature. Analysis of the binding affinity to thyroid hormones of recombinant human TTR showed a dissociation constant (Kd) for triiodothyronine (T3) of 53.26+/-3.97 nM and for thyroxine (T4) of 19.73+/-0.13 nM. These values are similar to those found for TTR purified from human serum, and gave a Kd T3/T4 ratio of 2.70. The affinity for T4 of human/crocTTR (Kd=22.75+/-1.89 nM) was higher than those of both human TTR and C. porosus TTR, but the affinity for T3 (Kd=5.40+/-0.25 nM) was similar to C. porosus TTR, giving a Kd T3/T4 ratio of 0.24. A similar affinity for both T3 (Kd=57.78+/-5.65 nM) and T4 (Kd=59.72+/-3.38 nM), with a Kd T3/T4 ratio of 0.97, was observed for truncated crocTTR. The obtained results strongly confirm the hypothesis that the unstructured N-terminal region of TTR critically influences the specificity and affinity of thyroid hormone binding to TTR.     

Monoaryl derivatives as transthyretin fibril formation inhibitors: Design,synthesis, biological evaluation and structural analysis

Transthyretin (TTR) is a ß-sheet-rich homotetrameric protein that transports thyroxine (T4) and retinol both in plasma and in cerebrospinal fluid. TTR also interacts with amyloid-β, playing a protective role in Alzheimer’s disease. Dissociation of the native transthyretin (TTR) tetramer is widely accepted as the critical step in TTR amyloids fibrillogenesis, and is responsible for extracellular deposition of amyloid fibrils. Small molecules, able to bind in T4 binding sites and stabilize the TTR tetramer, are interesting tools to treat and prevent systemic ATTR amyloidosis. We report here the synthesis, in vitro evaluation and three-dimensional crystallographic analyses of new monoaryl-derivatives in complex with TTR. Of the derivatives reported here, the best inhibitor of TTR fibrillogenesis, 1d, exhibits an activity similar to diflunisal.     

Evolution of structure, ontogeny of gene expression, and function of Xenopus laevis transthyretin

Xenopus laevis transthyretin (xTTR) cDNA was cloned and sequenced. The derived amino acid sequence was very similar to those of other vertebrate transthyretins (TTR). TTR gene expression was observed during metamorphosis in X. laevis tadpole liver but not in tadpole brain nor adult liver. Recombinant xTTR was synthesized in Pichia pastoris and identified by amino acid sequence, subunit molecular mass, tetramer formation, and binding to retinol-binding protein. Contrary to mammalian xTTRs, the affinity of xTTR was higher for L-triiodothyronine than for L-thyroxine. The regions of the TTR genes coding for the NH(2)-terminal sections of the polypeptide chains of TTR seem to have evolved by stepwise shifts of mRNA splicing sites between exons 1 and 2, resulting in shorter and more hydrophilic NH(2) termini. This may be one molecular mechanism of positive Darwinian evolution. Open reading frames with xTTR-like sequences in the genomes of C. elegans and several microorganisms suggested evolution of the TTR gene from ancestor TTR gene-like "DNA modules." Increasing preference for binding of L-thyroxine over L-triiodothyronine may be associated with evolving tissue-specific regulation of thyroid hormone action by deiodination.     

Tetramer dissociation and monomer partial unfolding precedes protofibril formation in amyloidogenic transthyretin variants

Amyloid fibril formation and deposition is a common feature of a wide range of fatal diseases including spongiform encephalopathies, Alzheimer's disease, and familial amyloidotic polyneuropathies (FAP), among many others. In certain forms of FAP, the amyloid fibrils are mostly constituted by variants of transthyretin (TTR), a homotetrameric plasma protein. Recently, we showed that transthyretin in solution may undergo dissociation to a non-native monomer, even under close to physiological conditions of temperature, pH, ionic strength, and protein concentration. We also showed that this non-native monomer is a compact structure, does not behave as a molten globule, and may lead to the formation of partially unfolded monomeric species and high molecular mass soluble aggregates (Quintas, A., Saraiva, M. J. M., and Brito, R. M. M. (1999) J. Biol. Chem. 274, 32943-32949). Here, based on aging experiments of tetrameric TTR and chemically induced protein unfolding experiments of the non-native monomeric forms, we show that tetramer dissociation and partial unfolding of the monomer precedes amyloid fibril formation. We also show that TTR variants with the least thermodynamically stable non-native monomer produce the largest amount of partially unfolded monomeric species and soluble aggregates under conditions that are close to physiological. Additionally, the soluble aggregates formed by the amyloidogenic TTR variants showed morphological and thioflavin-T fluorescence properties characteristic of amyloid. These results allowed us to conclude that amyloid fibril formation by some TTR variants might be triggered by tetramer dissociation to a compact non-native monomer with low conformational stability, which originates partially unfolded monomeric species with a high tendency for ordered aggregation into amyloid fibrils. Thus, partial unfolding and conformational fluctuations of molecular species with marginal thermodynamic stability may play a crucial role on amyloid formation in vivo.     

Kinetic stabilization of an oligomeric protein under physiological conditions demonstrated by a lack of subunit exchange: implications for transthyretin amyloidosis

Kinetic stabilization of transthyretin (TTR) is established to prevent human neurodegeneration. Therefore, small molecule-mediated kinetic stabilization of the native state is an attractive strategy to prevent the misfolding and misassembly associated with TTR amyloid disease. Since the physiological microenvironment resulting in human TTR amyloidogenesis remains unclear, the conservative approach is to identify inhibitors that function under a variety of conditions. Small molecule kinetic stabilization of TTR has been established by concentration-dependent inhibition of acid-mediated amyloidogenesis and urea-induced tetramer dissociation. Since denaturing conditions reduce the binding affinity of inhibitors making it difficult to predict inhibitor efficacy under physiological conditions, we introduce a method for quantifying kinetic stabilization under physiological conditions. The rate of subunit exchange between wild-type TTR homotetramers and wild-type TTR homotetramers tagged with an N-terminal acidic flag tag is dictated by the rate of tetramer dissociation to its monomeric subunits prior to reassembly, rendering this method ideally suited for assessing the kinetic stabilization of TTR imparted by small molecule binding and evaluating small molecule binding constants. Addition of amyloidogenesis inhibitors to this exchange reaction slows tetramer dissociation in a concentration-dependent manner, stopping dissociation at concentrations where at least one inhibitor is bound to each tetramer in solution. Subunit exchange enables the rate of tetramer dissociation and the kinetic stabilization imparted by small molecule binding to be evaluated under physiological conditions in which the TTR concentration is not reduced by aggregation or irreversible dissociation.     

Natural polyphenols inhibit different steps of the process of transthyretin (TTR) amyloid fibril formation

Several natural polyphenols with potent inhibitory effects on amyloid fibril formation have been reported. Herein, we studied modulation of transthyretin (TTR) fibrillogenesis by selected polyphenols. We demonstrate that both curcumin and nordihydroguaiaretic acid (NDGA) bind to TTR and stabilize the TTR tetramer. However, while NDGA slightly reduced TTR aggregation, curcumin strongly suppressed TTR amyloid fibril formation by generating small "off-pathway" oligomers and EGCG maintained most of the protein in a non-aggregated soluble form. This indicates alternative mechanisms of action supported by the occurrence of different non-toxic intermediates. Moreover, EGCG and curcumin efficiently disaggregated pre-formed TTR amyloid fibrils. Our studies, together with the safe toxicological profile of these phytochemicals may guide a novel pharmacotherapy for TTR-related amyloidosis targeting different steps in fibrillogenesis.     

Anion shielding of electrostatic repulsions in transthyretin modulates stability and amyloidosis: insight into the chaotrope unfolding dichotomy

The balance between stabilizing forces and the localized electrostatic repulsions destabilizing the transthyretin (TTR) tetramer is tunable via anion shielding. The two symmetrical anion interaction sites in TTR are comprised of residues Lys15 and Lys15' from opposing subunits on the periphery of the two thyroxine binding sites. These epsilon-ammonium groups repel one another and destabilize the tetramer, unless an appropriate anion is present, which stabilizes the tetramer. Chaotrope denaturation of TTR exhibits unusual behavior in that urea appears to be a stronger denaturant than GdmCl (guanidinium chloride), even though GdmCl is typically twice as powerful as a denaturant. The shift in the midpoint of the urea denaturation curve to higher concentrations as well as the increase in the mole fraction of tetramer that is highly resistant to denaturation with increasing KCl concentration provides strong evidence that anion shielding stabilizes the TTR tetramer. A consequence of tetramer stabilization is folding hysteresis, because the high GdmCl concentrations required to denature the anion-stabilized tetramer do not allow refolding of the unfolded monomers. The formation of amyloid fibrils by TTR requires that its normal tetrameric structure dissociate to alternatively folded monomers, a process mediated by acidification (pH 5-4). This process is inhibited by Cl(-) ions in a concentration-dependent fashion. Chloride ion may not be the relevant physiological TTR stability modulator, but it is the main focus of these studies explaining the hysteresis observed in the denaturation and refolding studies with GdmCl.     

Destabilised human transthyretin shapes the morphology of calcium carbonate crystals

Human transthyretin (TTR) is a homotetramer that transports thyroid hormones and retinol in the serum and cerebrospinal fluid. TTR is also an intracellular protein found in tissues such as those in the brain, eye and pancreas. TTR is a nutrition marker, reflecting the health of the organism, and TTR levels are linked to the normal and diseased states of the body. The switch from a protective to a pathological role is attributed to the destabilisation of the TTR structure, which leads to tetramer dissociation and amyloid formation. Native and destabilised TTR have been associated with osteoarthritis and bone density in humans. Moreover, TTR is present in eggshell mammillary cones; therefore, we verified the putative TTR engagement in the process of mineral formation. Using an in vitro assay, we found that TTR affected calcium carbonate crystal growth and morphology, producing asymmetric crystals with a complex nanocrystalline composition. The crystals possessed rounded edges and corners and irregular etch pits, suggesting the selective inhibition of crystal growth and/or dissolution imposed by TTR. The occurrence of many porosities, fibrillary inclusions and amorphous precipitates suggested that destabilisation of the TTR structure is an important factor involved in the mineralisation process. Crystals grown in the presence of TTR exhibited the characteristic features of crystals controlled by biomineralisation-active proteins, suggesting novel functions of TTR in the mineral formation process.     

Interaction of diethylstilbestrol and ioxynil with transthyretin in chicken serum

The association of suspected endocrine-disrupting chemicals (EDCs), diethylstilbestrol (DES), ioxynil and pentachlorophenol (PCP), with chicken serum proteins was investigated in relation to thyroid system disruption. All of these chemicals strongly inhibited l-[(125)I]thyroxine ([(125)I]T(4)) binding to purified transthyretin (TTR) whereas PCP was less potent inhibitor than DES and ioxynil of [(125)I]T(4) binding to diluted whole chicken serum. This result suggested that PCP interacted with serum proteins other than TTR in whole chicken serum. Following the incubation of chicken serum with each chemical (final concentrations 0.25-1.0 microM), serum proteins were fractionated by gel filtration chromatography (Cellulofine GCL-1000) and affinity chromatography (human retinol-binding protein coupled to Sepharose 4B). Although all chemicals were detected in the gel filtration chromatography 50-100 kDa fractions, DES and ioxynil, but not PCP, were co-eluted with TTR during affinity chromatography. Our results indicated that a significant proportion of DES and ioxynil, but a low proportion of PCP, interacted with TTR in whole chicken serum.