Transthyretin variants with improved inhibition of ?-amyloid aggregation.
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ABSTRACT:
SUBMITTER: Mangrolia P
PROVIDER: S-EPMC4867095 | biostudies-literature | 2016 Jun
REPOSITORIES: biostudies-literature
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Project description:β-Amyloid (Aβ) aggregation is causally linked to Alzheimer's disease. On the basis of in vitro and transgenic animal studies, transthyretin (TTR) is hypothesized to provide neuroprotection against Aβ toxicity by binding to Aβ and inhibiting its aggregation. TTR is a homotetrameric protein that circulates in blood and cerebrospinal fluid; its normal physiological role is as a carrier for thyroxine and retinol-binding protein (RBP). RBP forms a complex with retinol, and the holoprotein (hRBP) binds with high affinity to TTR. In this study, the role of TTR ligands in TTR-mediated inhibition of Aβ aggregation was investigated. hRBP strongly reduced the ability of TTR to inhibit Aβ aggregation. The effect was not due to competition between Aβ and hRBP for binding to TTR, as Aβ bound equally well to TTR-hRBP complexes and TTR. hRBP is known to stabilize the TTR tetrameric structure. We show that Aβ partially destabilizes TTR and that hRBP counteracts this destabilization. Taken together, our results support a mechanism wherein TTR-mediated inhibition of Aβ aggregation requires not only TTR-Aβ binding but also destabilization of TTR quaternary structure.
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Project description:Transthyretin (TTR) is a homotetrameric protein that is found in the plasma and cerebrospinal fluid. Dissociation of TTR tetramers sets off a downhill cascade of amyloid formation through polymerization of monomeric TTR. Interestingly, TTR has an additional, biologically relevant activity, which pertains to its ability to slow the progression of amyloid beta (Aβ) associated pathology in transgenic mice. In vitro, both TTR and a kinetically stable variant of monomeric TTR (M-TTR) inhibit the fibril formation of Aβ1-40/42 molecules. Published evidence suggests that tetrameric TTR binds preferentially to Aβ monomers, thus destabilizing fibril formation by depleting the pool of Aβ monomers from aggregating mixtures. Here, we investigate the effects of M-TTR on the in vitro aggregation of Aβ1-42 . Our data confirm previous observations that fibril formation of Aβ is suppressed in the presence of sub-stoichiometric amounts of M-TTR. Despite this, we find that sub-stoichiometric levels of M-TTR are not bona fide inhibitors of aggregation. Instead, they co-aggregate with Aβ to promote the formation of large, micron-scale insoluble, non-fibrillar amorphous deposits. Based on fluorescence correlation spectroscopy measurements, we find that M-TTR does not interact with monomeric Aβ. Two-color coincidence analysis of the fluorescence bursts of Aβ and M-TTR labeled with different fluorophores shows that M-TTR co-assembles with soluble Aβ aggregates and this appears to drive the co-aggregation into amorphous precipitates. Our results suggest that mimicking the co-aggregation activity with protein-based therapeutics might be a worthwhile strategy for rerouting amyloid beta peptides into inert, insoluble, and amorphous deposits.
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Project description:The protective effect of transthyretin (TTR) on cellular toxicity of β-amyloid (Aβ) has been previously reported. TTR is a tetrameric carrier of thyroxine in blood and cerebrospinal fluid, the pathogenic aggregation of which causes systemic amyloidosis. However, studies have documented a protective effect of TTR against cellular toxicity of pathogenic Aβ, a protein associated with Alzheimer's disease. TTR binds Aβ, alters its aggregation, and inhibits its toxicity both in vitro and in vivo In this study, we investigate whether the amyloidogenic ability of TTR and its antiamyloid inhibitory effect are associated. Using protein aggregation and cytotoxicity assays, we found that the dissociation of the TTR tetramer, required for its amyloid pathogenesis, is also necessary to prevent cellular toxicity from Aβ oligomers. These findings suggest that the Aβ-binding site of TTR may be hidden in its tetrameric form. Aided by computational docking and peptide screening, we identified a TTR segment that is capable of altering Aβ aggregation and toxicity, mimicking TTR cellular protection. EM, immune detection analysis, and assessment of aggregation and cytotoxicity revealed that the TTR segment inhibits Aβ oligomer formation and also promotes the formation of nontoxic, nonamyloid amorphous aggregates, which are more sensitive to protease digestion. Finally, this segment also inhibits seeding of Aβ catalyzed by Aβ fibrils extracted from the brain of an Alzheimer's patient. Together, these findings suggest that mimicking the inhibitory effect of TTR with peptide-based therapeutics represents an additional avenue to explore for the treatment of Alzheimer's disease.
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Project description:Alzheimer's disease (AD) is characterized by progressive neurodegeneration associated with amyloid β (Aβ) peptide aggregation. The aggregation of Aβ monomers (AβMs) leads to the formation of Aβ oligomers (AβOs), the neurotoxic Aβ form, capable of permeating the cell membrane. Here, we investigated the effect of a fluorene-based active drug candidate, named K162, on both Aβ aggregation and AβO toxicity toward the bilayer lipid membrane (BLM). Electrochemical impedance spectroscopy (EIS), atomic force microscopy (AFM), and molecular dynamics (MD) were employed to show that K162 inhibits AβOs-induced BLM permeation, thus preserving BLM integrity. In the presence of K162, only shallow defects on the BLM surface were formed. Apparently, K162 modifies Aβ aggregation by bypassing the formation of toxic AβOs, and only nontoxic AβMs, dimers (AβDs), and fibrils (AβFs) are produced. Unlike other Aβ toxicity inhibitors, K162 preserves neurologically beneficial AβMs. This unique K162 inhibition mechanism provides an alternative AD therapeutic strategy that could be explored in the future.
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Project description:Misfolding and amyloid formation of transthyretin (TTR) is implicated in numerous degenerative diseases. TTR misfolding is greatly accelerated under acidic conditions, and thus most of the mechanistic studies of TTR amyloid formation have been conducted at various acidic pH values (2-5). In this study, we report the effect of pH on TTR misfolding pathways and amyloid structures. Our combined solution and solid-state NMR studies revealed that TTR amyloid formation can proceed via at least two distinct misfolding pathways depending on the acidic conditions. Under mildly acidic conditions (pH 4.4), tetrameric native TTR appears to dissociate to monomers that maintain most of the native-like β-sheet structures. The amyloidogenic protein undergoes a conformational transition to largely unfolded states at more acidic conditions (pH 2.4), leading to amyloid with distinct molecular structures. Aggregation kinetics is also highly dependent upon the acidic conditions. TTR quickly forms moderately ordered amyloids at pH 4.4, while the aggregation kinetics is dramatically reduced at a lower pH of 2.4. The effect of the pathogenic mutations on aggregation kinetics is also markedly different under the two different acidic conditions. Pathogenic TTR variants (V30M and L55P) aggregate more aggressively than WT TTR at pH 4.4. In contrast, the single-point mutations do not affect the aggregation kinetics at the more acidic condition of pH 2.4. Given that the pathogenic mutations lead to more aggressive forms of TTR amyloidoses, the mildly acidic condition might be more suitable for mechanistic studies of TTR misfolding and aggregation.
| S-EPMC6339575 | biostudies-literature
Project description:Self-association of β-amyloid (Aβ) into soluble oligomers and fibrillar aggregates is associated with Alzheimer's disease pathology, motivating the search for compounds that selectively bind to and inhibit Aβ oligomerization and/or neurotoxicity. Numerous small-molecule inhibitors of Aβ aggregation or toxicity have been reported in the literature. However, because of their greater size and complexity, peptides and peptidomimetics may afford improved specificity and affinity as Aβ aggregation modulators compared to small molecules. Two divergent strategies have been employed in the search for peptides that bind Aβ: (i) using recognition domains corresponding to sequences in Aβ itself (such as KLVFF) and (ii) screening random peptide-based libraries. In this study, we propose a third strategy, specifically, designing peptides that mimic binding domains of Aβ-binding proteins. Transthyretin, a plasma transport protein that is also relatively abundant in cerebrospinal fluid, has been shown to bind to Aβ, inhibit aggregation, and reduce its toxicity. Previously, we identified strand G of transthyretin as a specific Aβ binding domain. In this work we further explore and define the necessary features of this binding domain. We demonstrate that peptides derived from transthyretin bind Aβ and inhibit its toxicity. We also show that, although both transthyretin and transthyretin-derived peptides bind Aβ and inhibit toxicity, they differ significantly in their effect on Aβ aggregation.
| S-EPMC4102968 | biostudies-literature
Project description:Transthyretin (TTR), a homotetrameric protein that transports thyroxine and retinol both in plasma and in cerebrospinal (CSF) fluid provides a natural protective response against Alzheimer's disease (AD), modulates amyloid-β (Aβ) deposition by direct interaction and co-localizes with Aβ in plaques. TTR levels are lower in the CSF of AD patients. Zn2+, Mn2+ and Fe2+ transform TTR into a protease able to cleave Aβ. To explain these activities, monomer dissociation or conformational changes have been suggested. Here, we report that when TTR crystals are exposed to copper or iron salts, the tetramer undergoes a significant conformational change that alters the dimer-dimer interface and rearranges residues implicated in TTR's ability to neutralize Aβ. We also describe the conformational changes in TTR upon the binding of the various metal ions. Furthermore, using bio-layer interferometry (BLI) with immobilized Aβ(1-28), we observe the binding of TTR only in the presence of copper. Such Cu2+-dependent binding suggests a recognition mechanism whereby Cu2+ modulates both the TTR conformation, induces a complementary Aβ structure and may participate in the interaction. Cu2+-soaked TTR crystals show a conformation different from that induced by Fe2+, and intriguingly, TTR crystals grown in presence of Aβ(1-28) show different positions for the copper sites from those grown its absence.
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Project description:Cistanche tubulosa aqueous extract (CTE) is already used as a botanical prescription drug for treating dementia in China. Our previous studies reported that phenylethanoid glycosides of CTE have anti-Alzheimer's disease (AD) activity by inhibiting amyloid β peptide (Aβ) aggregation and deposition. However, recent studies considered that the phenylethanoid glycosides may be metabolized by intestinal bacteria, because all analysis results showed that the bioavailability of phenylethanoid glycosides is extremely low. In this study we demonstrate how iron chelation plays a crucial role in the Aβ aggregation and deposition inhibition mechanism of phenylethanoid glycosides of CTE. In addition, we further proved phenylethanoid glycosides (1⁻3) could reach brain. Active CTE component and action mechanism confirmation will be a great help for product quality control and bioavailability studies in the future. At the same time, we provide a new analysis method useful in determining phenylethanoid glycosides (1⁻3) in plants, foods, blood, and tissues for chemical fingerprint and pharmacokinetic research.
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Project description:Alzheimer's disease is associated with the deposition of the amyloid-β peptide (Aβ) into extracellular senile plaques in the brain. In vitro and in vivo observations have indicated that transthyretin (TTR) acts as an Aβ scavenger in the brain, but the mechanism has not been fully resolved. We have monitored the aggregation process of Aβ40 by thioflavin T fluorescence, in the presence or absence of different concentrations of preformed seed aggregates of Aβ40, of wild-type tetrameric TTR (WT-TTR), and of a variant engineered to be stable as a monomer (M-TTR). Both WT-TTR and M-TTR were found to inhibit specific steps of the process of Aβ40 fibril formation, which are primary and secondary nucleations, without affecting the elongation of the resulting fibrils. Moreover, the analysis shows that both WT-TTR and M-TTR bind to Aβ40 oligomers formed in the aggregation reaction and inhibit their conversion into the shortest fibrils able to elongate. Using biophysical methods, TTR was found to change some aspects of its overall structure following such interactions with Aβ40 oligomers, as well as with oligomers of Aβ42, while maintaining its overall topology. Hence, it is likely that the predominant mechanism by which TTR exerts its protective role lies in the binding of TTR to the Aβ oligomers and in inhibiting primary and secondary nucleation processes, which limits both the toxicity of Aβ oligomers and the ability of the fibrils to proliferate.
| S-EPMC7997117 | biostudies-literature
Project description:Amyloid-formation by the islet amyloid polypeptide (IAPP), produced by the β-cells in the human pancreas, has been associated with the development of type II diabetes mellitus (T2DM). The human plasma-protein transthyretin (TTR), a well-known amyloid-inhibiting protein, is interestingly also expressed within the IAPP producing β-cells. In the present study, we have characterized the ability of TTR to interfere with IAPP amyloid-formation, both in terms of its intrinsic stability as well as with regard to the effect of TTR-stabilizing drugs. The results show that TTR can prolong the lag-phase as well as impair elongation in the course of IAPP-amyloid formation. We also show that the interfering ability correlates inversely with the thermodynamic stability of TTR, while no such correlation was observed as a function of kinetic stability. Furthermore, we demonstrate that the ability of TTR to interfere is maintained also at the low pH environment within the IAPP-containing granules of the pancreatic β-cells. However, at both neutral and low pH, the addition of TTR-stabilizing drugs partly impaired its efficacy. Taken together, these results expose mechanisms of TTR-mediated inhibition of IAPP amyloid-formation and highlights a potential therapeutic target to prevent the onset of T2DM.
| S-EPMC8001701 | biostudies-literature