Transthyretin Anti-Amyloidogenic and Fibril Disrupting Activities of Bacopa monnieri (L.) Wettst (Brahmi) Extract.

The homotetrameric plasma protein transthyretin (TTR), is responsible for a series of debilitating and often fatal disorders in humans known as transthyretin amyloidosis. Currently, there is no cure for TTR amyloidosis and treatment options are rare. Thus, the identification and development of effective and safe therapeutic agents remain a research imperative. The objective of this study was to determine the effectiveness of Bacopa monnieri extract (BME) in the modulation of TTR amyloidogenesis and disruption of preformed fibrils. Using aggregation assays and transmission electron microscopy, it was found that BME abrogated the formation of human TTR aggregates and mature fibrils but did not dis-aggregate pre-formed fibrils. Through acid-mediated and urea-mediated denaturation assays, it was revealed that BME mitigated the dissociation of folded human TTR and L55P TTR into monomers. ANS binding and glutaraldehyde cross-linking assays showed that BME binds at the thyroxine-binding site and possibly enhanced the quaternary structural stability of native TTR. Together, our results suggest that BME bioactives prevented the formation of TTR fibrils by attenuating the disassembly of tetramers into monomers. These findings open up the possibility of further exploration of BME as a potential resource of valuable anti-TTR amyloidosis therapeutic ingredients.

The catalytic kinetics of chicken transthyretin towards human Aß1-42.

The novel property of transthyretin (TTR) as a protease has been proposed to be significant. However, the study of TTR proteolysis properties has not been completely elucidated. Herein, we first report the catalytic activity of chicken TTR from plasma determined by using fluorescently labeled amyloid beta 1-42 peptide (Aß1-42), and compared it with human TTR (human TTR) from plasma and recombinant Crocodylus porosus TTR. The enzyme kinetic study revealed that the affinity for Aß1-42 of chicken TTR and C. porosus TTR (KM values were 12.72 ±â€¯0.27 µM and 16.21 ±â€¯0.02 µM, respectively) were significantly lower than human TTR (KM was 43.05 ±â€¯0.39 µM). In addition, the catalytic efficiency of chicken TTR (Kcat/KM was 310,386.87 ±â€¯13,627.12 M-1 s-1) was 4.3 and 5.5 folds higher than those of C. porosus TTR and human TTR (Kcat/KM were 72,893.80 ±â€¯355.74 M-1 s-1 and 56,519.12 ±â€¯5009.50 M-1 s-1, respectively), respectively. These results does not only indicated the relationship between structure and the proteolytic activity of TTR, but also suggested a potential development of TTR as a therapeutic anti-Aß agent.

Structural Stabilization of Human Transthyretin by Centella asiatica (L.) Urban Extract: Implications for TTR Amyloidosis.

Transthyretin is responsible for a series of highly progressive, degenerative, debilitating, and incurable protein misfolding disorders known as transthyretin (TTR) amyloidosis. Since dissociation of the homotetrameric protein to its monomers is crucial in its amyloidogenesis, stabilizing the native tetramer from dissociating using small-molecule ligands has proven a viable therapeutic strategy. The objective of this study was to determine the potential role of the medicinal herb Centella asiatica on human transthyretin (huTTR) amyloidogenesis. Thus, we investigated the stability of huTTR with or without a hydrophilic fraction of C. asiatica (CAB) against acid/urea-mediated denaturation. We also determined the influence of CAB on huTTR fibrillation using transmission electron microscopy. The potential binding interactions between CAB and huTTR was ascertained by nitroblue tetrazolium redox-cycling and 8-anilino-1-naphthalene sulfonic acid displacement assays. Additionally, the chemical profile of CAB was determined by liquid chromatography quadruple time-of-flight mass spectrometry (HPLC-QTOF-MS). Our results strongly suggest that CAB bound to and preserved the quaternary structure of huTTR in vitro. CAB also prevented transthyretin fibrillation, although aggregate formation was unmitigated. These effects could be attributable to the presence of phenolics and terpenoids in CAB. Our findings suggest that C. asiatica contains pharmaceutically relevant bioactive compounds which could be exploited for therapeutic development against TTR amyloidosis.

The hydrophobic C-terminal sequence of transthyretin affects its catalytic kinetics towards amidated neuropeptide Y.

Transthyretin (TTR) is a transporter for thyroid hormone and retinol binding protein that has recently been reported to have proteolytic activity against certain substrates, including amidated neuropeptide Y (NPY). However, the proteolytic activity of TTR towards NPY is not fully understood. Here, we used fluorescence-based assays to determine the catalytic kinetics of human TTR towards human amidated NPY. The Michaelis constant (KM) and catalytic efficiency (kcat/KM) of TTR proteolysis were 15.88 ± 0.44 µm and 687 081 ± 35 692 m -1·s-1, respectively. In addition, we demonstrated an effect of the C-terminal sequence of TTR. When the C-terminal sequence of TTR was made more hydrophobic, the KM and kcat/KM changed to 12.87 ± 0.22 µm and 983 755 ± 18 704 m -1·s-1, respectively. Our results may be useful for the development of TTR as a therapeutic agent with low risk of the undesirable symptoms that develop from amidated NPY, and for further improvement of the kcat/KM of TTR.

Increasing the length and hydrophobicity of the C-terminal sequence of transthyretin strengthens its binding affinity to retinol binding protein.

Transthyretin (TTR) is a transporter for thyroid hormone (TH) and retinol, the latter via binding with retinol binding protein (RBP). Both the N-terminal and C-terminal regions of the TTR subunit are located in close proximity to the central binding channel for ligands. During the evolution of vertebrates, these regions changed in length and hydropathy. The changes in the N-terminal sequence were demonstrated to affect the binding affinities for THs and RBP. Here, the effects of changes in the C-terminal sequence were determined. Three chimeric TTRs, namely pigC/huTTR (human TTR with the C-terminal sequence changed to that of Sus scrofa TTR), xenoN/pigC/huTTR (human TTR with the N-terminal and C-terminal sequences changed to those of Xenopus laevis and S. scrofa, respectively), and pigC/crocTTR (Crocodylus porosus TTR with the C-terminal sequence changed to that of S. scrofa TTR), were constructed and their binding affinities for human RBP were determined at low TTR/RBP molar ratio using chemiluminescence immunoblotting. The binding dissociation constant (Kd) values of pigC/huTTR, xenoN/pigC/huTTR and pigC/crocTTR were 3.20 ± 0.35, 1.53 ± 0.38 and 0.31 ± 0.04 µm, respectively, and the Kd values of human and C. porosus TTR were 4.92 ± 0.68 and 1.42 ± 0.45 µm, respectively. These results demonstrate chimeric TTRs bound RBP with a higher strength than wild-type TTRs, and the changes in the C-terminal sequence of TTR had a positive effect on its binding affinity for RBP. In addition, changes to the N-terminal and C-terminal sequences showed comparable effects on the binding affinity.

Gene structure and evolution of transthyretin in the order Chiroptera.

Bats are mammals in the order Chiroptera. Although many extensive morphologic and molecular genetics analyses have been attempted, phylogenetic relationships of bats has not been completely resolved. The paraphyly of microbats is of particular controversy that needs to be confirmed. In this study, we attempted to use the nucleotide sequence of transthyretin (TTR) intron 1 to resolve the relationship among bats. To explore its utility, the complete sequences of TTR gene and intron 1 region of bats in Vespertilionidae: genus Eptesicus (Eptesicus fuscus) and genus Myotis (Myotis brandtii, Myotis davidii, and Myotis lucifugus), and Pteropodidae (Pteropus alecto and Pteropus vampyrus) were extracted from the retrieved sequences, whereas those of Rhinoluphus affinis and Scotophilus kuhlii were amplified and sequenced. The derived overall amino sequences of bat TTRs were found to be very similar to those in other eutherians but differed from those in other classes of vertebrates. However, missing of amino acids from N-terminal or C-terminal region was observed. The phylogenetic analysis of amino acid sequences suggested bat and other eutherian TTRs lineal descent from a single most recent common ancestor which differed from those of non-placental mammals and the other classes of vertebrates. The splicing of bat TTR precursor mRNAs was similar to those of other eutherian but different from those of marsupial, bird, reptile and amphibian. Based on TTR intron 1 sequence, the inferred evolutionary relationship within Chiroptera revealed more closely relatedness of R. affinis to megabats than to microbats. Accordingly, the paraphyly of microbats was suggested.

Effect of the N-terminal sequence on the binding affinity of transthyretin for human retinol-binding protein.

During vertebrate evolution, the N-terminal region of transthyretin (TTR) subunit has undergone a change in both length and hydropathy. This was previously shown to change the binding affinity for thyroid hormones (THs). However, it was not known whether this change affects other functions of TTR. In the present study, the effect of these changes on the binding of TTR to retinol-binding protein (RBP) was determined. Two wild-type TTRs from human and Crocodylus porosus, and three chimeric TTRs, including a human chimeric TTR in which its N-terminal sequence was changed to that of C. porosus TTR (croc/huTTR) and two C. porosus chimeric TTRs (hu/crocTTR in which its N-terminal sequence was changed to that of human TTR and xeno/crocTTR in which its N-terminal sequence was changed to that of Xenopus laevis TTR), were analyzed for their binding to human RBP by native-PAGE followed by immunoblotting and a chemilluminescence assay. The K(d) of human TTR was 30.41 ± 2.03 µm, and was similar to that reported for the second binding site, whereas that of crocodile TTR was 2.19 ± 0.24 µm. The binding affinities increased in croc/huTTR (K(d) = 23.57 ± 3.54 µm) and xeno/crocTTR (K(d) = 0.61 ± 0.16 µm) in which their N-termini were longer and more hydrophobic, but decreased in hu/crocTTR (K(d) = 5.03 ± 0.68 µm) in which its N-terminal region was shorter and less hydrophobic. These results suggest an influence of the N-terminal primary structure of TTR on its function as a co-carrier for retinol with RBP.

In vitro thyroid hormone-disrupting activity in effluents and surface waters in Thailand.

The thyroid hormone (TH)-disrupting activity of effluents and environmental water samples in Thailand was surveyed by three in vitro bioassays with different endpoints. These assays test the potency of competitive binding with the active form of TH, 3,3',5-[(125)I]triiodo-l-thyronine (T(3)), to the plasma transport protein transthyretin (TTR) and TH receptor (TR; the TTR assay and TR assay, respectively) and the interference with the cellular T(3)-signaling pathway through TR-mediated luciferase gene activation (the luc assay). The TH-disrupting activity in water samples collected from paper manufacturing plants (PMPs), the canal Khlong U-Taphao, and a sewage-treatment plant (STP) was detected predominantly in the dichloromethane/methanol or methanol fractions of solid-phase extraction, suggesting a similar hydrophobic nature of the causative contaminants. The TR assay was relatively more sensitive than the TTR assay to the competitively potent contaminants. The luc assay indicated that the dichloromethane/methanol fractions of most water samples contained anti-T(3)-like activity. Our assays demonstrated that wastewater treatment effectively removed the TH-disrupting contaminants from wastewater in the PMP and the STP. The potencies for TH disruption at the three sampling points of the STP exhibited positive correlations among the three bioassays, whereas those from the canal and PMP water were not correlated among the three bioassays. Furthermore, the influent contaminants that were competitively potent in the TTR assay partially affected the luc assay. These bioassays are useful monitoring tools that give results relevant for evaluating the health of amphibian populations.

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.

Crocodile transthyretin: structure, function, and evolution.

Structure and function were studied for Crocodylus porosus transthyretin (crocTTR), an important intermediate in TTR evolution. The cDNA for crocTTR mRNA was cloned and sequenced and the amino acid sequence of crocTTR was deduced. In contrast to mammalian TTRs, but similar to avian and lizard TTRs, the subunit of crocTTR had a long and hydrophobic NH(2)-terminal region. Different from the situation in mammals, triiodothyronine (T(3)) was bound by crocTTR with higher affinity than thyroxine (T(4)). Recombinant crocTTR and a chimeric construct, with the NH(2)-terminal region of crocTTR being replaced by that of Xenopus laevis TTR, were synthesized in the yeast Pichia pastoris. Analysis of the affinity of the chimeric TTRs showed that the NH(2)-terminal region modulates T(4) and T(3) binding characteristics of TTR. The structural differences of the NH(2)-terminal regions of reptilian and amphibian TTRs were caused by a shift in splice sites at the 5' end of exon 2. The comparison of crocodile and other vertebrate TTRs shows that TTR evolution is an example for positive Darwinian evolution and identifies its molecular mechanism.