Synthesis of an analog of the thyroid hormone-binding protein transthyretin via regioselective chemical ligation

Transthyretin is an essential protein responsible for the transport of thyroid hormones and retinol in human serum and is also implicated in the amyloid diseases familial amyloidotic polyneuropathy and senile systemic amyloidosis. Its folding properties and stabilization by ligands are of current interest due to their importance in understanding and combating these diseases. Here we report the solid phase synthesis of the monomeric unit of a transthyretin analog (equivalent to 127 amino acids) using t-Boc chemistry and peptide ligation and its folding to form a functional 54-kDa tetramer. The monomeric unit of the protein was chemically synthesized in three parts (positions 1--51, 54--99, and 102--127) and ligated using a chemoselective thioether ligation chemistry. The synthetic protein was folded and assembled to a tetrameric structure in the presence of transthyretin's native ligand, thyroxine, as shown by gel filtration chromatography, native gel electrophoresis, transthyretin antibody recognition, and thyroid hormone binding. Other folding products included a high molecular weight aggregate as well as a transient dimeric species. This represents one of the largest macromolecules chemically synthesized to date and demonstrates the potential of protein chemical synthesis for investigations of protein-ligand interactions.     

Application of an allosteric model to describe the interactions among retinol binding protein 4, transthyretin, and small molecule retinol binding protein 4 ligands

Retinol binding protein 4 (RBP4) is a serum protein that serves as the major transport protein for retinol (vitamin A). Recent reports suggest that elevated levels of RBP4 are associated with insulin resistance and that insulin sensitivity may be improved by reducing serum RBP4 levels. This can be accomplished by administration of small molecules, such as fenretinide, that compete with retinol for binding to RBP4 and disrupt the protein-protein interaction between RBP4 and transthyretin (TTR), another serum protein that protects RBP4 from renal clearance. We developed a fluorescence resonance energy transfer (FRET) assay that measures the interaction between RBP4 and TTR and can be used to determine the binding affinities of RBP4 ligands. We present an allosteric model that describes the pharmacology of interaction among RBP4, TTR, retinol, and fenretinide, and we show data that support the model. We show that retinol increases the affinity of RBP4 for TTR by a factor of 4 and determine the affinity constants of fenretinide and retinyl acetate. The assay may be useful for characterizing small molecule ligands that bind to RBP4 and disrupt its interaction with TTR. In addition, such a model could be used to describe other protein-protein interactions that are modulated by small molecules.     

Consideration on some hormone binding proteins patterns during pregnancy

Serum concentrations of sex hormone binding globulin, transcortin, thyroxine binding globulin, transthyretin together with retinol binding protein, ceruloplasmin, transferrin and albumin were measured sequentially in pregnant women in order to derive more definite suppositions relating to the prime function of hormone binding proteins. Thus, the fact that except for transthyretin all other specific hormone binding proteins exhibited appreciable but significantly variable increases would suggest: a) the apparent existence of more complex mechanisms regulating protein metabolism during pregnancy than hitherto postulated (i.e. the general notion of an integrated estrogen influence); b) a major and distinctive role for each of the hormone binding proteins is plausible since alterations in hormonal requirements by the fetus as pregnancy progresses can not be provided by the almost constant transplacental transfer rate of the "free" hormone moiety.     

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.     

Vitamin A transport: in vitro models for the study of RBP secretion

Retinol-binding protein (RBP) is the specific plasma carrier of retinol, encharged of the vitamin transport from the liver to target cells. Ligand binding influences the RBP affinity for transthyretin (TTR), a homotetrameric protein involved in the RBP/TTR circulating complex, and the secretion rate of RBP. In fact, in vitamin A deficiency, the RBP release from the hepatocytes dramatically decreases and the protein accumulates in the cells, until retinol is available again. The mechanism is still not clear and new cellular models are needed to understand in detail how the soluble RBP can be retained inside the cell. In fish, a vitamin A transport system similar to that of higher vertebrates is emerging, although with significant differences.     

Delineation of in vivo assembled multiprotein complexes via biomolecular interaction analysis mass spectrometry

Biomolecular interaction analysis mass spectrometry (BIA-MS) is a multiplexed bioanalytical approach used in analysis of proteins from complex biological mixtures. It utilizes surface-immobilized ligands for protein affinity retrieval, surface plasmon resonance for monitoring the ligand-protein interaction and matrix-assisted laser desorption/ionization-time of flight mass spectrometry for revealing the masses of the biomolecules retrieved by the ligand. In order to explore the utility of BIA-MS in delineation of multiprotein complexes, an in vivo assembled protein complex comprised of retinol binding protein (RBP) and transthyretin (TTR) was investigated. Antibodies to RBP and TTR were utilized as ligands in the analysis of the protein complex present in human plasma. The RBP-TTR complex was retrieved by the anti-RBP antibody as indicated by the presence of both RBP and TTR signals in the mass spectra. RBP signals were not observed in the mass spectra of the material retained on the anti-TTR derivatized surface. In addition, the mass-specific detection in BIA-MS allowed detection of RBP and TTR analyte variants.     

HINT predictive analysis of binding between retinol binding protein and hydrophobic ligands

The interaction between the retinol binding protein and four ligands was evaluated using HINT, a software based on experimental LogP values of individual atoms. A satisfactory correlation was found between the HINT scores and the experimental dissociation constants of three of the ligands, fenretinide, N-ethylretinamide and all-trans retinol, despite their hydrophobic nature. A prediction is made for the binding affinity of the fourth ligand, axerophtene, not yet determined in solution.     

The absence of transthyretin does not impair regulation of lipid and glucose metabolism.

Increased levels of neuropeptide Y have been reported in transthyretin-null mice. This effect might be related to transthyretin ligands (retinol and thyroxine) since, through binding to nuclear receptors, they modulate the expression of genes that control cellular metabolism. The retinoic X receptors form obligatory heterodimers with peroxisome proliferator-activated receptors and liver X receptors - potent regulators of fat, glucose and cholesterol homeostasis. We used transthyretin-null mice to investigate whether the absence of transthyretin influences metabolism. Transthyretin-null mice do not differ from controls in body weight and white adipose tissue morphology, nor in basal or fast-induced circulating levels of glucose, lipids, and leptin. Glucose tolerance tests show that transthyretin-null mice have normal capacity to remove and metabolize energy substrates. Expression of genes encoding lipid transporters and nuclear receptors are also similar in transthyretin-null and control mice. Therefore, the absence of transthyretin does not seem to influence the regulation of lipid and glucose metabolism.     

视黄醇结合蛋白的结构与功能

视黄醇结合蛋白（RBP）是视黄醇转运的载体蛋白,作为结合小分子流水物质的载体蛋白家族（lipocalin）的一个重要成员,其结构与功能的研究正受到国外学者的重视,文章介绍了视黄醇结合蛋白的性质、结构研究进展,讨论了视黄醇结合蛋白与前蛋白和受体相互作用的位点和结构特点．     

Cross talk between signaling and vitamin A transport by the retinol-binding protein receptor STRA6

The plasma membrane protein STRA6 transports vitamin A from its blood carrier retinol binding protein (RBP) into cells, and it also functions as a cytokine receptor which activates JAK/STAT signaling. We show here that, unlike other cytokine receptors, phosphorylation of STRA6 is not simply induced upon binding of its extracellular ligand. Instead, activation of the receptor is triggered by STRA6-mediated translocation of retinol from serum RBP to an intracellular acceptor, the retinol-binding protein CRBP-I. The observations also demonstrate that the movement of retinol from RBP to CRBP-I, and thus activation of STRA6, is critically linked to the intracellular metabolism of the vitamin. Furthermore, the data show that STRA6 phosphorylation is required for retinol uptake to proceed. Hence, the observations demonstrate that STRA6 orchestrates a multicomponent "machinery" that couples vitamin A homeostasis and metabolism to activation of a signaling cascade and that, in turn, STRA6 signaling regulates the cellular uptake of the vitamin. STRA6 appears to be a founding member of a new class of proteins that may be termed "cytokine signaling transporters."     

Ligand-dependent secretion of rat retinol-binding protein expressed in HeLa cells.

A minigene encoding rat retinol-binding protein (RBP) was transfected into HeLa cells, which do not express endogenous RBP, transthyretin, or cellular retinol-binding protein. The HeLa cells manufactured and secreted the transfected gene product, demonstrating that RBP-transthyretin assembly is not a requirement for the secretion of RBP. When HeLa cells were grown under vitamin A-deficient conditions, RBP accumulated in the endoplasmic reticulum. Both serum and retinol stimulated secretion of RBP in a concentration-dependent manner. The retinol-regulated secretion occurred also after protein synthesis had been blocked by cycloheximide. Addition of holo-RBP or retinal, but not retinoic acid, stimulated secretion of RBP. Thus, an in vitro model system that resembles the rat hepatocyte in vivo with regard to the known regulation of RBP secretion has been established in a human cell line of extrahepatic origin. It can be concluded that cellular retinol-binding protein is not required for the transfer of retinol to RBP and that the mechanism whereby retinol controls the intracellular transport of RBP is neither specific for tissues synthesizing RBP nor species-specific. To investigate the structural properties responsible for the endoplasmic reticulum retention of RBP in the absence of its ligand, a cDNA encoding chicken purpurin, a protein that is 50% identical to RBP and that binds retinol, was expressed in HeLa cells. In contrast to RBP, purpurin was not retained in vitamin A-deficient HeLa cells.     

Receptor-mediated uptake and internalization of transthyretin

Evidence of cellular transthyretin (TTR) binding was sought because of the observation that transthyretin can increase the uptake of its hormonal ligand. Transthyretin was bound by human hepatoma (Hep G2) cells in a time- and temperature-dependent manner, reaching equilibrium within 2 h. Scatchard analysis was consistent with a single class of high affinity binding sites with a Kd of approximately 5 nM at 0 and 4 degrees C and 14 nM at 37 degrees C. These dissociation constants are more than 2 orders of magnitude lower than the concentration of transthyretin in human serum. The apparent capacity at 0 degrees C, corrected for internalized TTR, was approximately 20,000 sites/cell. Saturable, high affinity binding of human transthyretin was also demonstrable with rat primary hepatocytes and human renal adenocarcinoma, neuroblastoma, and transformed lung cells. Rat and human transthyretin were equipotent in displacing isotopically labeled, species-specific transthyretin from human hepatoma cells and rat primary hepatocytes, a finding that is consistent with the strong homology between rat and human transthyretin. Eighty-eight percent of the saturable uptake was internalized as determined by proteolytic removal of surface transthyretin. Internalization was dependent on receptor binding and was more markedly inhibited than surface binding at 0 degrees C. Concentrations of thyroxine within a range that saturated a significant proportion of the primary and secondary TTR iodothyronine binding sites increased the uptake and internalization of transthyretin in a dose-dependent manner. By analogy to the function of receptors for other transport proteins, the interaction between transthyretin and its receptor is likely to affect ligand delivery and may have additional metabolic effects.     

Delineating protein-protein interactions via biomolecular interaction analysis-mass spectrometry

The utility of biomolecular interaction analysis-mass spectrometry (BIA/MS) in screening for protein-protein interactions was explored in this work. Experiments were performed in which proteins served as ligands for screening of possible interactions with other proteins from human plasma and urine. The proteins utilized were beta-2-microglobulin, cystatin C (cysC), retinol binding protein (RBP), transthyretin (TTR), alpha-1-microglobulin, C-reactive protein, transferrin and papain. The immobilization of functionally active proteins was confirmed via interactions with antibodies to the corresponding proteins. Various dilutions of human urine and plasma were injected over the protein-derivatized surfaces. It was observed that the urine injections generally yielded smaller SPR responses than those observed after the plasma injections. The BIA/MS experiments did not reveal novel protein-protein interactions, although several established interactions (such as those between RBP and TTR, and cysC and papain) were validated. Few protein ligand deficiencies (such as truncations) leading to false negative and false positive BIA/MS results were also discovered.     

Molecular dynamics simulation study of AG10 and tafamidis binding to the Val122Ile transthyretin variant

Molecular dynamics (MD) simulations were used to investigate the binding of four ligands to the Val122Ile mutant of the protein transthyretin. Dissociation, misfolding, and subsequent aggregation of mutated transthyretin proteins are associated with the disease Familial Amyloidal Cardiomyopathy. The ligands investigated were the drug candidate AG10 and its decarboxy and N-methyl derivatives along with the drug tafamidis. These ligands bound to the receptor in two halogen binding pockets (HBP) designated AB and A’B’. Inter-ligand distances, solvent accessible surface areas, root mean squared deviation measurements, and extracted structures showed very little change in the AG10 ligands' conformations or locations within the HBP during the MD simulation. In addition, the AG10 ligands experienced stable, two-point interactions with the protein by forming hydrogen bonds with Ser-117 residues in both the AB and A’B’ binding pockets and Lysine-15 residues found near the surface of the receptor. Distance measurements showed these H-bonds formed simultaneously during the MD simulation. Removal of the AG10 carboxylate functional group to form decarboxy-AG10 disrupted this two-point interaction causing the ligand in the AB pocket to undergo a conformational change during the MD simulation. Likewise, addition of a methyl group to the AG10 hydrazone functional group also disrupted the two-point interaction by decreasing hydrogen bonding interactions with the receptor. Finally, MD simulations showed that the tafamidis ligands experienced fewer hydrogen bonding interactions than AG10 with the protein receptor. The tafamidis ligand in pocket A’B’ was also found to move deeper into the HBP during the MD simulation.     

Characterization of the molten globule state of retinol-binding protein using a molecular dynamics simulation approach

Retinol-binding protein transports retinol, and circulates in the plasma as a macromolecular complex with the protein transthyretin. Under acidic conditions retinol-binding protein undergoes a transition to the molten globule state, and releases the bound retinol ligand. A biased molecular dynamics simulation method has been used to generate models for the ensemble of conformers populated within this molten globule state. Simulation conformers, with a radius of gyration at least 1.1 A greater than that of the native state, contain on average 37%beta-sheet secondary structure. In these conformers the central regions of the two orthogonal beta-sheets that make up the beta-barrel in the native protein are highly persistent. However, there are sizable fluctuations for residues in the outer regions of the beta-sheets, and large variations in side chain packing even in the protein core. Significant conformational changes are seen in the simulation conformers for residues 85-104 (beta-strands E and F and the E-F loop). These changes give an opening of the retinol-binding site. Comparisons with experimental data suggest that the unfolding in this region may provide a mechanism by which the complex of retinol-binding protein and transthyretin dissociates, and retinol is released at the cell surface.     

Comparison of the modelled thyroxine binding site in TBG with the experimentally determined site in transthyretin.

The structure of cleaved thyroxine-binding globulin (TBG) has been modelled on the crystal structure of cleaved alpha 1-antitrypsin (a member of the serine proteinase inhibitor, serpin, superfamily) based on the high sequence homology exhibited by the two proteins. Particular attention was paid to the identification and modelled characteristics of the thyroxine binding site. The primary aim of the study was to compare the site qualitatively with the crystallographically determined binding site of transthyretin, the other major transporter of thyroxine, in an attempt to explain the higher binding affinity of the site compared with the known thyroxine binding site in transthyretin (10(10) versus 10(8) M-1). The proposed binding site shares some similar characteristics with the transthyretin binding site but also includes a cluster of aromatic residues which are entirely absent in transthyretin. It is proposed that this might account for the substantial difference in binding affinities.     

Vitamins A and E in digestive cancers

Vitamins A and E, transthyretin (TT) and retinol binding protein (RBP) plasma levels were assayed in 70 patients having a recently diagnosed digestive cancer and in 116 controls. After adjustment for sex and age, vitamin A or RBP and TT were found lowered in digestive cancer but vitamin E was lowered only in esophagus cancer. The relationship of vitamin levels to the relative risk of digestive cancer was calculated by multiple logistic conditional regression. TT level remained the only significant parameter. Since TT levels are strongly influenced by the nutritional status of patients, our results suggest that the decreased blood levels of vitamin A and its carriers observed in digestive cancer are the consequence of nutritional alterations evoked by the disease.     

Interactions of retinol with binding proteins: studies with retinol-binding protein and with transthyretin.

The interactions within the molecular complex in which retinol circulates in blood were studied. To monitor binding between retinol-binding protein (RBP) and transthyretin (TTR), TTR was labeled with a long-lived fluorescence probe (pyrene). Changes in the rotational volume of TTR following its association with RBP were monitored by fluorescence anisotropy of the probe. Titration of TTR with holo-RBP revealed the presence of 1.5 binding sites characterized by a dissociation constant Kd = 0.07 microM. At 0.15 M NaCl, binding of RBP to TTR showed an absolute requirement for the native ligand, retinol. At higher ionic strength (0.5 M NaCl), RBP complexed with retinal also bound to TTR with high affinity (Kd = 0.134 microM). RBP containing retinoic acid did not bind to TTR even at the high salt concentration. The data suggest that the TTR binding site on RBP is in close proximity to the retinoid binding site and that the head group of retinoic acid, when bound to RBP, presents steric hindrance for the interactions with TTR. The implications of the data for selectivity in retinoid transport in the circulation are discussed. The kinetics of the steps leading to complete dissociation of the retinol-RBP-TTR complex was also studied. The first step of this process was dissociation of retinol, which had a rate constant of 0.06/min. Following loss of retinol, the two proteins dissociate. The rate of dissociation is slow (k = 0.055/h), however, indicating that the complex apo-RBP-TTR will be an important factor in regulating serum levels of retinol.     

The ligand-binding site of bovine beta-lactoglobulin: evidence for a function?

Ever since the fortuitous observation that beta-lactoglobulin (beta-Lg), the major whey protein in the milk of ruminants, bound retinol, the details of the binding have been controversial. beta-Lg is a lipocalin, like plasma retinol-binding protein, so that ligand association was expected to make use of the central cavity in the protein. However, an early crystallographic analysis and some of the more recent solution studies indicated binding elsewhere. We have now determined the crystal structures of the complexes of the trigonal form of beta-Lg at pH 7.5 with bound retinol (R=21.4% for 7329 reflections between 20 and 2.4 A resolution, R(free)=30.6%) and with bound retinoic acid (R=22.7% for 7813 reflections between 20 and 2.34 A resolution, R(free)=29.8%). Both ligands are found to occupy the central calyx in a manner similar to retinol binding in retinol-binding protein. We find no evidence of binding at the putative external binding site in either of these structural analyses. Further, competition between palmitic acid and retinol reveals only palmitate bound to the protein. An explanation is provided for the lack of ligand binding to the orthorhombic crystal form also obtained at pH 7.5. Finally, the possible function of beta-Lg is discussed in the light of its species distribution and similarity to other lipocalins.     

Molecular docking studies on interaction of diverse retinol structures with human alcohol dehydrogenases predict a broad role in retinoid ligand synthesis.

Some members of the human alcohol dehydrogenase (ADH) family possess retinol dehydrogenase activity and may thus function in production of the active nuclear receptor ligand retinoic acid. Many diverse natural forms of retinol exist including all-trans-retinol (vitamin A(1)), 9-cis-retinol, 3,4-didehydroretinol (vitamin A(2)), 4-oxo-retinol, and 4-hydroxy-retinol as well as their respective carboxylic acid derivatives which are active ligands for retinoid receptors. This raises the question of whether ADHs can accommodate all these different retinols and thus participate in the activation of several retinoid ligands. The crystal structures of human ADH1B and ADH4 provide the opportunity to examine their active sites for potential binding to many diverse retinol structures using molecular docking algorithms. The criteria used to score successful docking included achievement of distances of 1.9-2.4 A between the catalytic zinc and the hydroxyl oxygen of retinol and 3.2-3.6 A between C-4 of the coenzyme NAD and C-15 of retinol. These distances are sufficient to enable hydride transfer during the oxidation of an alcohol to an aldehyde. By these criteria, all-trans-retinol, 4-oxo-retinol, and 4-hydroxy-retinol were successfully docked to both ADH1B and ADH4. However, 9-cis-retinol and 3,4-didehydroretinol, which have more restrictive conformations, were successfully docked to only ADH4 which possesses a wider active site than ADH1B and more easily accommodates the C-19 methyl group. Furthermore, docking of all retinols was more favorable in the active site of ADH4 rather than ADH1B as measured by force field and contact scores. These findings suggest that ADH1B has a limited capacity to metabolize retinols, but that ADH4 is well suited to function in the metabolism of many diverse retinols and is predicted to participate in the synthesis of the active ligands all-trans-retinoic acid, 9-cis-retinoic acid, 3, 4-didehydroretinoic acid, 4-oxo-retinoic acid, and 4-hydroxy-retinoic acid.