Retention of misfolded mutant transthyretin by the chaperone BiP/GRP78 mitigates amyloidogenesis

Carriers of the D18G transthyretin (TTR) mutation display an unusual central nervous system (CNS) phenotype with late onset of disease. D18G TTR is monomeric and highly prone to misfold and aggregate even at physiological conditions. Extremely low levels of mutant protein circulate both in human serum and cerebrospinal fluid, indicating impaired secretion of D18G TTR. Recent data show efficient selective ER-associated degradation (ERAD) of D18G TTR. One essential component of the ER-assisted folding machinery is the molecular chaperone BiP. Co-expression of BiP and D18G TTR, or BiP and wild-type (wt) TTR, or mutants A25T TTR and L55P TTR in Escherichia coli showed that only D18G TTR was significantly captured by BiP. Negligible capture of wt TTR and L55P TTR was seen and a sixfold smaller amount of A25T TTR bound to BiP compared to D18G TTR. These data correlate very well with thermodynamic and kinetic stability of the TTR variants, indicating that folding efficiency is inversely correlated to BiP capture. The complexes between BiP and D18G TTR were stable and could be isolated through affinity chromatography. Analytical ultracentrifugation and size-exclusion chromatography revealed that D18G TTR and BiP formed a mixture of 1:1 complexes and large soluble oligomers. The stoichiometry of captured D18G TTR versus BiP increased with increasing size of the oligomers. This indicates that BiP either worked as a molecular shepherd collecting the aggregation-prone mutant into stable oligomers or that BiP could bind to oligomers formed from misfolded mutant protein. Sequence analysis of bound TTR peptides to BiP revealed a bound sequence corresponding to residues 88-103 of TTR, comprising beta-strand F in the folded TTR monomer constituting part of the hydrogen bonding tetramer interface in native TTR. The F-strand has also been suggested as a possible elongation region of amyloid fibrils, implicating how substoichiomeric amounts of BiP could sequester prefibrillar amyloidogenic oligomers through binding to this part of TTR. BiP binding to D18G TTR was abolished by addition of ATP. The released D18G TTR completely misfolded into amyloid aggregates as shown by ThT fluorescence and Congo red binding.     

High resolution crystal structures of piscine transthyretin reveal different binding modes for triiodothyronine and thyroxine

Transthyretin (TTR) is an extracellular transport protein involved in the distribution of thyroid hormones and vitamin A. So far, TTR has only been found in vertebrates, of which piscine TTR displays the lowest sequence identity with human TTR (47%). Human and piscine TTR bind both thyroid hormones 3,5,3'-triiodo-l-thyronine (T(3)) and 3,5,3',5'-tetraiodo-l-thyronine (thyroxine, T(4)). Human TTR has higher affinity for T(4) than T(3), whereas the reverse holds for piscine TTR. X-ray structures of Sparus aurata (sea bream) TTR have been determined as the apo-protein at 1.75 A resolution and bound to ligands T(3) and T(4), both at 1.9 A resolution. The apo structure is similar to human TTR with structural changes only at beta-strand D. This strand forms an extended loop conformation similar to the one in chicken TTR. The piscine TTR.T(4) complex shows the T(4)-binding site to be similar but not identical to human TTR, whereas the TTR.T(3) complex shows the I3' halogen situated at the site normally occupied by the hydroxyl group of T(4). The significantly wider entrance of the hormone-binding channel in sea bream TTR, in combination with its narrower cavity, provides a structural explanation for the different binding affinities of human and piscine TTR to T(3) and T(4).     

Comparative studies of two transthyretin variants with protective effects on familial amyloidotic polyneuropathy: TTR R104H and TTR T119M

Recently, a new nonpathogenic transthyretin (TTR) variant-TTR R104H (TTR H104)-has been described in heterozygotic and compound heterozygotic individuals from a Japanese family with familial amyloidotic polyneuropathy (FAP). The compound heterozygotic individual, a carrier of TTR V30M (TTR M30) and TTR R104H (TTR M30/H104) presented a very mild form of FAP with slow progression of the disease. TTR and retinol binding protein (RBP) levels were found to be increased in serum from TTR H104 carriers. These characteristics are very similar to those found in compound heterozygotic carriers of TTR V30M-T119M (TTR M30/M119). To structurally compare these variants, we performed stability and thyroxine (T(4)) binding studies. TTR M30/H104 showed an increased resistance to dissociation into monomers similar to TTR M30/M119. This suggests that the His104 substitution has the same stabilizing effect on tetrameric TTR as the Met119 substitution. Concerning T(4) binding, TTR H104 presents a T(4) binding affinity lower than that of TTR M119, but still higher than normal TTR. However, TTR from the compound heterozygotic carrier of TTR M30/H104 presented a T(4) binding affinity lower than normal. The results indicate that the His 104 substitution induces structural alterations that increase the stability of the tetramer in compound heterozygotes for TTR M30 despite a lower affinity for T(4) binding. Thus, stability of TTR and binding affinity for T(4) may not be related. More detailed characterization of these variants is needed to clarify the structural alterations responsible for their increased stability.     

Quantification of the thermodynamically linked quaternary and tertiary structural stabilities of transthyretin and its disease-associated variants: the relationship between stability and amyloidosis

Urea denaturation studies were carried out as a function of transthyretin (TTR) concentration to quantify the thermodynamically linked quaternary and tertiary structural stability and to improve our understanding of the relationship between mutant folding energetics and amyloid disease phenotype. Urea denaturation of TTR involves at least two equilibria: dissociation of tetramers into folded monomers and monomer unfolding. To deal with the thermodynamic linkage of these equilibria, we analyzed concentration-dependent denaturation data by globally fitting them to an equation that simultaneously accounts for the two-step denaturation process. Using this method, the quaternary and tertiary structural stabilities of well-behaved TTR sequences, wild-type (WT) TTR and the disease-associated variant V122I, were scrutinized. The V122I variant is linked to late onset familial amyloid cardiomyopathy, the most common familial TTR amyloid disease. V122I TTR exhibits a destabilized quaternary structure and a stable tertiary structure relative to those of WT TTR. Three other variants of TTR were also examined, L55P, V30M, and A25T TTR. The L55P mutation is associated with the most aggressive familial TTR amyloid disease. L55P TTR has a complicated denaturation pathway that includes dimers and trimers, so globally fitting its concentration-dependent urea denaturation data yielded error-laden estimates of stability parameters. Nevertheless, it is clear that L55P TTR is substantially less stable than WT TTR, primarily because its tertiary structure is unstable, although its quaternary structure is destabilized as well. V30M is the most common mutation associated with neuropathic forms of TTR amyloid disease. V30M TTR is certainly destabilized relative to WT TTR, but like L55P TTR, it has a complex denaturation pathway that cannot be fit to the aforementioned two-step denaturation model. Literature data suggest that V30M TTR has stable quaternary structure but unstable tertiary structure. The A25T mutant, associated with central nervous system amyloidosis, is highly aggregation-prone and exhibits drastically reduced quaternary and tertiary structural stabilities. The observed differences in stability among the disease-associated TTR variants highlight the complexity and heterogeneity of TTR amyloid disease, an observation that has important implications for the treatment of these maladies.     

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.     

D18G transthyretin is monomeric,aggregation prone,and not detectable in plasma and cerebrospinal fluid: a prescription for central nervous system amyloidosis?

Over 70 transthyretin (TTR) mutations facilitate amyloidosis in tissues other than the central nervous system (CNS). In contrast, the D18G TTR mutation in individuals of Hungarian descent leads to CNS amyloidosis. D18G forms inclusion bodies in Escherichia coli, unlike the other disease-associated TTR variants overexpressed to date. Denaturation and reconstitution of D18G from inclusion bodies afford a folded monomer that is destabilized by 3.1 kcal/mol relative to an engineered monomeric version of WT TTR. Since TTR tetramer dissociation is typically rate limiting for amyloid formation, the monomeric nature of D18G renders its amyloid formation rate 1000-fold faster than WT. It is perplexing that D18G does not lead to severe early onset systemic amyloidosis, given that it is the most destabilized TTR variant characterized to date, more so than variants exhibiting onset in the second decade. Instead, CNS impairment is observed in the fifth decade as the sole pathological manifestation; however, benign systemic deposition is also observed. Analysis of heterozygote D18G patient's serum and cerebrospinal fluid (CSF) detects only WT TTR, indicating that D18G is either rapidly degraded postsecretion or degraded within the cell prior to secretion, consistent with its inability to form hybrid tetramers with WT TTR. The nondetectable levels of D18G TTR in human plasma explain the absence of an early onset systemic disease. CNS disease may result owing to the sensitivity of the CNS to lower levels of D18G aggregate. Alternatively, or in addition, we speculate that a fraction of D18G made by the choroid plexus can be transiently tetramerized by the locally high thyroxine (T(4)) concentration, chaperoning it out into the CSF where it undergoes dissociation and amyloidogenesis due to the low T(4) CSF concentration. Selected small molecule tetramer stabilizers can transform D18G from a monomeric aggregation-prone state to a nonamyloidogenic tetramer, which may prove to be a useful therapeutic strategy against TTR-associated CNS amyloidosis.     

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.     

Study on the mechanism of interference of 3,4,3',4'-tetrachlorobiphenyl with the plasma retinol-binding proteins in rodents

The mechanism of plasma retinol reduction in rodents by 3,4,3',4'-tetrachlorobiphenyl (TCB) was investigated by radioimmunochemical analysis of the amounts of circulating and hepatic retinol-binding protein (RBP) and transthyretin (TTR) in exposed and control animals. Plasma RBP concentrations were markedly reduced in C57BL/Rij mice (50%) at 4 days, in DBA/2 mice (37-41%) at 4 and 8 days, and in Sprague-Dawley rats (58%) at 2 days after exposure to TCB. These reductions paralleled the time course of reduction of plasma retinol after exposure to TCB. Hepatic RBP concentrations were somewhat increased in TCB-treated animals, especially in the C57BL/Rij mouse and Sprague-Dawley rat. However, the release of hepatic RBP into the circulation was not blocked by TCB treatment, as analysed in vitamin A deficient rats. In addition, the amount of plasma TTR was in the normal range in TCB-treated rats. The dissociation constants of the RBP-TTR complex as analysed by polarization of fluorescence appeared to be significantly increased (from 0.5 x 10(-7) M-1 to 2.4 x 10(-7) M-1) in the presence of a TCB metabolite, isolated from plasma of TCB-treated rats. In addition, the estimated number of binding sites for RBP on the TTR molecule was reduced (from 2.8 to 1.7 sites) upon treatment of TTR with the TCB metabolite. These data support the hypothesis that plasma retinol reduction by TCB might result from a weakening of the RBP-TTR complex, in the presence of the TCB metabolite bound to the TTR.     

Interaction of chlorinated phenols with thyroxine binding sites of human transthyretin, albumin and thyroid binding globulin

Previous results (Brouwer and van den Berg, Toxicol. Appl. Pharmacol., 85 (1986) 301) indicated preferential binding of a hydroxylated metabolite of tetrachlorobiphenyl to transthyretin (TTR) a carrier of thyroxine (T4). In the present study it was investigated whether the T4 binding site of TTR could be occupied specifically by hydroxylated chlorinated aromatic compounds using chlorinated phenol congeners as model compounds in a competition assay with [125I]T4. Chlorinated aromatics such as 2,3-dichlorobenzene and 3,4,3',4'-tetrachlorobiphenyl, and phenols such as 4-hydroxybiphenyl and phenol were inefficient competitors. All chlorinated phenols tested were competitors for the T4 binding site of TTR. The ranking in competition was pentachlorophenol (PCP) greater than trichlorophenols greater than dichlorophenols greater than monochlorophenols. Structures with chlorine in both ortho positions to the hydroxyl group were more efficient competitors. The relative affinity of binding of pentachlorophenol (PCP) to TTR was about twice that of T4. Scatchard analysis showed that PCP mainly decreased the affinity constant K11 while the binding capacity R1 was not altered, indicating a competitive type of inhibition. PCP was also able to compete with T4 sites on albumin with a relative affinity of 0.25. T4 binding to thyroid binding globulin (TBG) was much less affected by interference of PCP (relative affinity 0.001). The results indicate a specific interaction of chlorophenols with the T4 binding site of TTR.     

Role of transthyretin in thyroxine transfer from cerebrospinal fluid to brain and choroid plexus

The transport of 125I-labeled thyroxine (T4) from the cerebrospinal fluid (CSF) into brain and choroid plexus (CP) was measured in anesthetized rabbit [0.5 mg/kg medetomidine (Domitor) and 10 mg/kg pentobarbitonal sodium (Sagatal) iv] using the ventriculocisternal (V-C) perfusion technique. 125I-labeled T4 contained in artificial CSF was continually perfused into the lateral ventricles for up to 4 h and recovered from the cisterna magna. The %recovery of 125I-labeled T4 from the aCSF was 47.2+/-5.6% (n=10), indicating removal of 125I-labeled T4 from the CSF. The recovery increased to 53.2+/-6.3% (n=4) and 57.8+/-14.8% (n=3), in the presence of 100 and 200 microM unlabeled-T4, respectively (P<0.05), indicating a saturable component to T4 removal from CSF. There was a large accumulation of 125I-labeled T4 in the CP, and this was reduced by 80% in the presence of 200 microM unlabeled T4, showing saturation. In the presence of the thyroid-binding protein transthyretin (TTR), more 125I-labeled T4 was recovered from CSF, indicating that the binding protein acted to retain T4 in CSF. However, 125I-labeled T4 uptake into the ependymal region (ER) of the frontal cortex also increased by 13 times compared with control conditions. Elevation was also seen in the hippocampus (HC) and brain stem. Uptake was significantly inhibited by the presence of endocytosis inhibitors nocodazole and monensin by >50%. These data suggest that the distribution of T4 from CSF into brain and CP is carrier mediated, TTR dependent, and via RME. These results support a role for TTR in the distribution of T4 from CSF into brain sites around the ventricular system, indicating those areas involved in neurogenesis (ER and HC).     

Potentially amyloidogenic conformational intermediates populate the unfolding landscape of transthyretin: Insights from molecular dynamics simulations

Protein aggregation into insoluble fibrillar structures known as amyloid characterizes several neurodegenerative diseases, including Alzheimer's, Huntington's and Creutzfeldt‐Jakob. Transthyretin (TTR), a homotetrameric plasma protein, is known to be the causative agent of amyloid pathologies such as FAP (familial amyloid polyneuropathy), FAC (familial amyloid cardiomiopathy) and SSA (senile systemic amyloidosis). It is generally accepted that TTR tetramer dissociation and monomer partial unfolding precedes amyloid fibril formation. To explore the TTR unfolding landscape and to identify potential intermediate conformations with high tendency for amyloid formation, we have performed molecular dynamics unfolding simulations of WT‐TTR and L55P‐TTR, a highly amyloidogenic TTR variant. Our simulations in explicit water allow the identification of events that clearly discriminate the unfolding behavior of WT and L55P‐TTR. Analysis of the simulation trajectories show that (i) the L55P monomers unfold earlier and to a larger extent than the WT; (ii) the single α‐helix in the TTR monomer completely unfolds in most of the L55P simulations while remain folded in WT simulations; (iii) L55P forms, early in the simulations, aggregation‐prone conformations characterized by full displacement of strands C and D from the main β‐sandwich core of the monomer; (iv) L55P shows, late in the simulations, severe loss of the H‐bond network and consequent destabilization of the CBEF β‐sheet of the β‐sandwich; (v) WT forms aggregation‐compatible conformations only late in the simulations and upon extensive unfolding of the monomer. These results clearly show that, in comparison with WT, L55P‐TTR does present a much higher probability of forming transient conformations compatible with aggregation and amyloid formation.     

Transthyretin protects against A-beta peptide toxicity by proteolytic cleavage of the peptide: a mechanism sensitive to the Kunitz protease inhibitor

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the deposition of amyloid beta-peptide (A-Beta) in the brain. Transthyretin (TTR) is a tetrameric protein of about 55 kDa mainly produced in the liver and choroid plexus of the brain. The known physiological functions of TTR are the transport of thyroid hormone T(4) and retinol, through binding to the retinol binding protein. TTR has also been established as a cryptic protease able to cleave ApoA-I in vitro. It has been described that TTR is involved in preventing A-Beta fibrilization, both by inhibiting and disrupting A-Beta fibrils, with consequent abrogation of toxicity. We further characterized the nature of the TTR/A-Beta interaction and found that TTR, both recombinant or isolated from human sera, was able to proteolytically process A-Beta, cleaving the peptide after aminoacid residues 1, 2, 3, 10, 13, 14,16, 19 and 27, as determined by mass spectrometry, and reversed phase chromatography followed by N-terminal sequencing. A-Beta peptides (1-14) and (15-42) showed lower amyloidogenic potential than the full length counterpart, as assessed by thioflavin binding assay and ultrastructural analysis by transmission electron microscopy. A-Beta cleavage by TTR was inhibited in the presence of an alphaAPP peptide containing the Kunitz Protease Inhibitor (KPI) domain but not in the presence of the secreted alphaAPP derived from the APP isoform 695 without the KPI domain. TTR was also able to degrade aggregated forms of A-Beta peptide. Our results confirmed TTR as a protective molecule in AD, and prompted A-Beta proteolysis by TTR as a protective mechanism in this disease. TTR may prove to be a useful therapeutic agent for preventing or retarding the cerebral amyloid plaque formation implicated in AD pathology.     

Evidence for the role of megalin in renal uptake of transthyretin

The kidney is a major organ for uptake of the thyroid hormone thyroxine (T(4)) and its conversion to the active form, triiodothyronine. In the plasma, one of the T(4) carriers is transthyretin (TTR). In the present study we observed that TTR, the transporter of both T(4) and retinol-binding protein, binds to megalin, the multiligand receptor expressed on the luminal surface of various epithelia including the renal proximal tubules. In the kidney, megalin plays an important role in tubular uptake of macromolecules filtered through the glomerulus. To evaluate the importance of megalin for renal uptake of TTR, we performed binding/uptake assays using immortalized rat yolk sac cells with high expression levels of megalin. Radiolabeled TTR, free as well as in complex with thyroxine or retinol-binding protein, was rapidly taken up by the cells, and the uptake was strongly inhibited by a polyclonal megalin antibody and by the receptor-associated protein, a chaperone-like protein inhibiting ligand binding to megalin. In cell culture, different TTR mutations presented different levels of cell association and degradation, suggesting that the structure of TTR is important for megalin recognition. Both the apo form and the T(4)-bound form were taken up by the cells. Analysis of urine from patients with Dent's disease, a renal tubular disorder that alters receptor-mediated endocytic reabsorption of proteins, identified TTR as an abundant excreted protein. Furthermore, analysis of kidney sections of megalin-deficient mice revealed no immunohistochemical TTR labeling in intracellular vesicles in the proximal tubule cells when compared with wild type control littermates. Taken together, the present data indicate that TTR represents a novel megalin ligand of importance in the thyroid hormone homeostasis.     

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.     

Chromium(III) ion and thyroxine cooperate to stabilize the transthyretin tetramer and suppress in vitro amyloid fibril formation

Transthyretin (TTR) amyloid fibril formation, which is triggered by the dissociation of tetrameric TTR, appears to be the causative factor in familial amyloidotic polyneuropathy and senile systemic amyloidosis. Binding of thyroxine (T(4)), a native ligand of TTR, stabilizes the tetramer, but the bioavailability of T(4) for TTR binding is limited due to the preferential binding of T(4) to globulin, the major T(4) carrier in plasma. Here, we show that Cr(3+) increased the T(4)-binding capacity of wild-type (WT) and amyloidogenic V30M-TTR. Moreover, we demonstrate that Cr(3+) and T(4) cooperatively suppressed in vitro fibril formation due to the stabilization of WT-TTR and V30M-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.     

Altered Circulating Levels of Retinol Binding Protein 4 and Transthyretin in Relation to Insulin Resistance,Obesity, and Glucose Intolerance in Asian Indians

Objective: Retinol binding protein 4 (RBP4) has been implicated in metabolic disorders including type 2 diabetes mellitus (T2DM), but few studies have looked at transthyretin (TTR) with which RBP4 is normally bound to in the circulation. We report on the systemic levels of RBP4 and TTR and their associations with insulin resistance, obesity, prediabetes, and T2DM in Asian Indians.Methods: Age-matched individuals with normal glucose tolerance (NGT, n = 90), impaired glucose tolerance (IGT, n = 70) and T2DM (n = 90) were recruited from the Chennai Urban Rural Epidemiology Study (CURES). Insulin resistance was estimated using the homeostasis model assessment of insulin resistance (HOMA-IR). RBP4 and TTR levels were measured by enzyme-linked immunosorbent assay (ELISA).Results: Circulatory RBP4 and TTR levels (in μg/mL) were highest in T2DM (RBP4: 13 ± 3.9, TTR: 832 ± 310) followed by IGT (RBP4: 10.5 ± 3.2; TTR: 720 ± 214) compared to NGT (RBP4: 8.7 ± 2.5; TTR: 551 ± 185; P<.001). Compared to nonobese NGT individuals, obese NGT, nonobese T2DM, and obese T2DM had higher RBP4 (8.1 vs. 10.6, 12.1, and 13.2 μg/mL, P<.01) and TTR levels (478 vs. 737, 777, and 900 μg/mL, P<.01). RBP4 but not TTR was significantly (P<.001) correlated with insulin resistance even among NGT subjects. In regression analysis, RBP4 and TTR showed significant associations with T2DM after adjusting for confounders (RBP4 odds ratio [OR]: 1.107, 95% confidence interval [CI]: 1.008–1.216; TTR OR: 1.342, 95% CI: 1.165–1.547).Conclusion: Circulatory levels of RBP4 and TTR showed a significant associations with glucose intolerance, obesity, T2DM and RBP4 additionally, with insulin resistance.Abbreviations: BMI = body mass index CI = confidence interval HDL = high-density lipoprotein IGT = impaired glucose tolerance LDL = low-density lipoprotein NGT = normal glucose tolerance OGTT = oral glucose tolerance test OR = odds ratio RBP4 = retinol binding protein 4 T2DM = type 2 diabetes mellitus TTR = transthyretin WC = waist circumference     

Production of recombinant human transthyretin with biological activities toward the understanding of the molecular basis of familial amyloidotic polyneuropathy (FAP).

Transthyretin (TTR) is a plasma protein interacting with thyroxine T4 and retinol binding protein (RBP). Several variants of TTR with single amino acid substitutions have been identified as the major components of the amyloid fibrils of familial amyloidotic polyneuropathy (FAP), a fetal, autosomal dominant genetic disease. The elucidation of the molecular nature of the variants distinct from that of the wild-type TTR is crucial for understanding the amyloidogenesis in FAP, but our understanding is very poor mainly because of the unavailability of pure variant TTRs. In the present study, we used an Escherichia coli OmpA secretion vector (Ghrayeb et al., 1984) and achieved an effective production of the variant TTRs related to FAP including Met-30, Ile-33, Ala-60, Tyr-77, Met-111, and Ile-122 types. The variant TTRs produced in this system were efficiently secreted to the culture media. The chemical analysis showed that the secreted TTR (Met-30 type) has the same N-terminus as the native one. IEF analyses also indicated that the secreted product is properly processed as assessed by its pI. Furthermore, the secreted TTR was shown to have biological activities, namely, the thyroxin binding activity and the ability to associate with retinol binding protein, indicating that the secreted TTR polypeptide is properly folded. The present work also demonstrated that the processing/secretion of the recombinant TTR molecules in E. coli was strongly affected by single amino acid substitutions.     

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.     

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.