Tetrameric Transthyretin Research Articles

Tetrameric Transthyretin as a Protective Factor Against Alzheimer's Disease.

Transthyretin (TTR) is a tetrameric protein traditionally recognized for its role in transporting thyroxine and retinol. Recent research has highlighted the potential neuroprotective functions of TTR in the setting of Alzheimer's disease (AD), which is the most common form of dementia and is caused by the deposition of amyloid beta (Aβ) and the resulting cytotoxic effects. This paper explores the mechanisms of TTR protective action, including its interaction with Aβ to prevent fibril formation and promote Aβ clearance from the brain. It also synthesizes experimental evidence suggesting that enhanced TTR stability may mitigate neurodegeneration and cognitive decline in AD. Potential therapeutic strategies such as small molecule stabilizers of TTR are discussed, highlighting their role in enhancing TTR binding to Aβ and facilitating its clearance. By consolidating current knowledge and proposing directions for future research, this review aims to underscore the significance of TTR as a neuroprotective factor in AD and the potential implications for future research.

Gene therapy options for hereditary transthyretin-related amyloidosis

Hereditary transthyretin-related amyloidosis (ATTRv) is arare autosomal dominant disease and is fatal if left untreated. It is caused by mutations in the transthyretin gene. All known mutations induce misfolding of the tetrameric transthyretin molecule and protein deposits in multiple organs. In peripheral nerves this result in sensorimotor and autonomic polyneuropathy and in cardiac muscle it causes cardiomyopathy. Untreated ATTRv is characterized by an irreversible and progressive course and death 7-11years after symptom onset. Treatment options consist of TTR stabilizing drugs, such as tafamidis and active agents that selectively interfere at the mRNA level, the so-called gene silencers patisiran and inotersen. All forms of treatment aim to prevent amyloid tissue deposition in tissues and organ dysfunction. Patisiran works by RNA interference on endogenous mechanisms of gene expression. It results in the cleavage of TTR-mRNA using the cytoplasmatic RNA-induced silencing complex. Inotersen, an antisense oligonucleotide, degrades TTR-mRNA via activation of nuclear RNaseH. Both mechanisms result in asignificant reduction of TTR protein serum levels. The efficacy could be demonstrated by slowing or improving neuropathy progression in the phase III study APOLLO (patisiran) or the NEURO-TTR study (inotersen). Furthermore, the use of both agents led to an improvement in the quality of life in patients with ATTRv. Both forms of treatment are approved in Germany since August 2018 for polyneuropathy stages1 and2 according to Coutinho. The choice of treatment should be carried out individually considering drug formulation, contraindications and the necessary safety monitoring controls.

Association of Transthyretin Val122Ile Variant With Incident Heart Failure Among Black Individuals

A genetic variant in the TTR gene (rs76992529; Val122Ile), present more commonly in individuals with African ancestry (population frequency: 3%-4%), causes misfolding of the tetrameric transthyretin protein complex that accumulates as extracellular amyloid fibrils and results in hereditary transthyretin amyloidosis. To estimate the association of the amyloidogenic Val122Ile TTR variant with the risk of heart failure and mortality in a large, geographically diverse cohort of Black individuals. Retrospective population-based cohort study of 7514 self-identified Black individuals living in the US participating in the REGARDS (Reasons for Geographic and Racial Differences in Stroke) study with genetic data available and without heart failure at baseline. The participants were enrolled at the baseline visit (2003-2007). The end of follow-up for the majority of outcomes was on December 31, 2018. All-cause mortality data were available through December 31, 2020. TTR Val122Ile (rs76992529) genotype. The primary outcome was incident heart failure (first hospitalization for heart failure or death due to heart failure). The secondary outcomes were heart failure mortality, cardiovascular mortality, and all-cause mortality. The multivariable Cox proportional hazards regression analyses were adjusted for genetic ancestry and demographic, clinical, and social factors. Among 7514 Black participants (median age, 64 years [IQR, 57-70 years]; 61% women), the population frequency of the TTR Val122Ile variant was 3.1% (232 variant carriers and 7282 noncarriers). During a median follow-up of 11.1 years (IQR, 5.9-13.5 years), incident heart failure occurred in 535 individuals (34 variant carriers and 501 noncarriers) and the incidence of heart failure was 15.64 per 1000 person-years among variant carriers vs 7.16 per 1000 person-years among noncarriers (adjusted hazard ratio [HR], 2.43 [95% CI, 1.71-3.46]; P < .001). Deaths due to heart failure occurred in 141 individuals (13 variant carriers and 128 noncarriers) and the incidence of heart failure mortality was 6.11 per 1000 person-years among variant carriers vs 1.85 per 1000 person-years among noncarriers (adjusted HR, 4.19 [95% CI, 2.33-7.54]; P < .001). Deaths due to cardiovascular causes occurred in 793 individuals (34 variant carriers and 759 noncarriers) and the incidence of cardiovascular death was 15.18 per 1000 person-years among variant carriers vs 10.61 per 1000 person-years among noncarriers (adjusted HR, 1.69 [95% CI, 1.19-2.39]; P = .003). Deaths due to any cause occurred in 2715 individuals (100 variant carriers and 2615 noncarriers) and the incidence of all-cause mortality was 41.46 per 1000 person-years among variant carriers vs 33.94 per 1000 person-years among noncarriers (adjusted HR, 1.46 [95% CI, 1.19-1.78]; P < .001). There was no significant interaction between TTR variant carrier status and sex on incident heart failure and the secondary outcomes. Among a cohort of Black individuals living in the US, being a carrier of the TTR Val122Ile variant was significantly associated with an increased risk of heart failure.

A quinoline derived D-A-D type fluorescent probe for sensing tetrameric transthyretin

Nowadays, with an upward trend in the prevalence of intracerebral amyloidosis, it is of great significance to use fluorescent probes for early diagnosis in vitro. In this study, a quinoline-derived D-A-D type chemosensor was rationally designed and synthesized as a probe for the sensitive detection of tetrameric transthyretin (WT-TTR).

Destabilisation of the structure of transthyretin is driven by Ca2+

Tetrameric transthyretin (TTR) transports thyroid hormones and retinol in plasma and cerebrospinal fluid and performs protective functions under stress conditions. Ageing and mutations result in TTR destabilisation and the formation of the amyloid deposits that dysregulate Ca2+ homeostasis. Our aim was to determine whether Ca2+ affects the structural stability of TTR. We show, using multiple techniques, that Ca2+ does not induce prevalent TTR dissociation and/or oligomerisation. However, in the presence of Ca2+, TTR exhibits altered conformational flexibility and different interactions with the solvent molecules. These structural changes lead to the formation of the sub-populations of non-native TTR conformers and to the destabilisation of the structure of TTR. Moreover, the sub-population of TTR molecules undergoes fragmentation that is augmented by Ca2+. We postulate that Ca2+ constitutes the structural and functional switch between the native and non-native forms of TTR, and therefore tip the balance towards age-dependent pathological calcification.

Optimization of kinetic stabilizers of tetrameric transthyretin: A prospective ligand efficiency-guided approach

In the past few years, attempts have been made to use decision criteria beyond Lipinski’s guidelines (Rule of five) to guide drug discovery projects more effectively. Several variables and formulations have been proposed and investigated within the framework of multiparameter optimization methods to guide drug discovery. In this context, the combination of Ligand Efficiency Indices (LEI) has been predominantly used to map and monitor the drug discovery process in a retrospective fashion. Here we provide an example of the use of a novel application of the LEI methodology for prospective lead optimization by using the transthyretin (TTR) fibrillogenesis inhibitor iododiflunisal (IDIF) as example. Using this approach, a number of compounds with theoretical efficiencies higher than the reference compound IDIF were identified. From this group, ten compounds were selected, synthesized and biologically tested. Half of the compounds (5, 6, 7, 8 and 10) showed potencies in terms of IC50 inhibition of TTR aggregation equal or higher than the lead compound. These optimized compounds mapped within the region of more efficient candidates in the corresponding experimental nBEI-NSEI plot, matching their position in the theoretical optimization plane that was used for the prediction. Due to their upstream (North-Eastern) position in the progression lines of NPOL = 3 or 4 of the nBEI-NSEI plot, three of them (5, 6 and 8) are more interesting candidates than iododiflunisal because they have been optimized in the three crucial LEI variables of potency, size and polarity at the same time. This is the first example of the effectiveness of using the combined LEIs within the decision process to validate the application of the LEI formulation for the prospective optimization of lead compounds.

Controlling Dissociation Channels of Gas-Phase Protein Complexes Using Charge Manipulation

Coarse-grained simulations with charge hopping were performed for a positively charged tetrameric transthyretin (TTR) protein complex with a total charge of +20. Charges were allowed to move among basic amino acid sites as well as N-termini. Charge distributions and radii of gyration were calculated for complexes simulated at two temperatures, 300 and 600K, under different scenarios. One scenario treated the complex in its normal state allowing charge to move to any basic site. Another scenario blocked protonation of all the N-termini except one. A final scenario used the complex in its normal state but added a basic-site containing tether (charge tag) near the N-terminus of one chain. The differences in monomer unfolding and charging were monitored in all three scenarios and compared. The simulation results show the importance of the N-terminus in leading the unfolding of the monomer units; a process that follows a zipper-like mechanism. Overall, experimentally modifying the complex by adding a tether or blocking the protonation of N-termini may give the potential for controlling the unraveling and subsequent dissociation of protein complexes.

The Diflunisal Trial: Study accrual and drug tolerance

Familial amyloidotic polyneuropathy (FAP) is a protein folding disorder that induces neuropathy and cardiomyopathy, leading to death within 7–15 years after onset of clinical disease. In vitro, small ligands binding the thyroid hormone docking site stabilize tetrameric transthyretin, inhibiting amyloid fibril formation. We undertook a randomized, placebo-controlled clinical trial to determine whether diflunisal, a well-known non-steroidal anti-inflammatory drug (NSAID) alters neurologic disease progression in FAP. We enrolled 130 subjects with wide age and FAP mutation representation. To date, few recognized complications of NSAIDs have occurred in the study cohort. Data collection will be completed by November 2012.

Gas-Phase Unfolding and Disassembly Reveals Stability Differences in Ligand-Bound Multiprotein Complexes

Mass spectrometry (MS) is widely used to assess the binding of small molecules to proteins and their complexes. In many cases, subtle differences in the stability afforded by binding of ligands to protein assemblies cannot be detected by MS. Here we show that monitoring the unfolding of protein subunits, using ion mobility-MS, allows differentiation of the effects of ligand binding not normally observed by MS alone. Using wild-type and disease-associated variants of tetrameric transthyretin, MS data indicate that populations of the variant protein are less stable than wild-type. Ion mobility-MS, however, is able to show that the natural ligand of transthyretin, thyroxine, provides a larger stability increase to the tetramer composed of variant subunits than to the wild-type protein-ligand complex. Overall, therefore, our results have implications for small-molecule drug design directed at multiprotein targets.

Prefibrillar transthyretin oligomers and cold stored native tetrameric transthyretin are cytotoxic in cell culture

Recent studies suggest that soluble, oligomeric species, which are intermediates in the fibril formation process in amyloid disease, might be the key species in amyloid pathogenesis. Soluble oligomers of human wild type transthyretin (TTR) were produced to elucidate oligomer properties. Employing ThT fluorescence, time-resolved fluorescence anisotropy of pyrene-labeled TTR, chemical cross-linking, and electron microscopy we demonstrated that early formed soluble oligomers (within minutes) from A-state TTR comprised on the average 20–30 TTR monomers. When administered to neuroblastoma cells these early oligomers proved highly cytotoxic and induced apoptosis after 48h of incubation. More mature fibrils (>24h of fibrillation) were non-toxic. Surprisingly, we also found that native tetrameric TTR, when purified and stored under cold conditions (4°C) was highly cytotoxic. The effect could be partially restored by increasing the temperature of the protein. The cytotoxic effects of native tetrameric TTR likely stems from a hitherto unexplored low temperature induced rearrangement of the tetramer conformation that possibly is related to the conformation of misfolded TTR in amyloigogenic oligomers.

Ion Mobility–Mass Spectrometry Reveals Long‐Lived, Unfolded Intermediates in the Dissociation of Protein Complexes

Folded or not? Ion mobility–mass spectrometry investigation of an activated macromolecular protein complex lends insight into the structures of intermediates formed in the dissociation process. The activated ions of human tetrameric transthyretin populate partially folded intermediate states (see picture; folded subunits in blue, partially unfolded subunits in red) prior to dissociation. Supporting information for this article is available on the WWW under http://www.wiley-vch.de/contents/jc_2002/2007/z702161_s.pdf or from the author. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Kinetic Assay for High-Throughput Screening of In Vitro Transthyretin Amyloid Fibrillogenesis Inhibitors

Stabilization of tetrameric transthyretin (TTR) by binding of small ligands is a current strategy aimed at inhibiting amyloid fibrillogenesis in transthyretin-associated pathologies, such as senile systemic amyloidosis (SSA) and familial amyloidotic polyneuropathy (FAP). A kinetic assay is developed for rapid evaluation of compounds as potential in vitro inhibitors in a high-throughput screening format. It is based on monitoring the time-dependent increase of absorbance due to turbidity occurring by acid-induced protein aggregation. The method uses the highly amyloidogenic Y78F mutant of human transthyretin (heterogously expressed in Escherichia coli cells). Initial rates of protein aggregation at different inhibitor concentrations follow a monoexponential dose-response curve from which inhibition parameters are calculated. For the assay development, thyroid hormones and nonsteroidal antiinflamatory drugs were chosen among other reference compounds. Some of them are already known to be in vitro inhibitors of TTR amyloidogenesis. Analysis time is optimized to last 1.5 h, and the method is implemented in microtiter plates for screening of libraries of potential fibrillogenesis inhibitors.

Screening Transthyretin Amyloid Fibril Inhibitors: Characterization of Novel Multiprotein, Multiligand Complexes by Mass Spectrometry

Tetrameric transthyretin is involved in transport of thyroxine and, through its interactions with retinol binding protein, vitamin A. Dissociation of these structures is widely accepted as the first step in the formation of transthyretin amyloid fibrils. Using a mass spectrometric approach, we have examined a series of 18 ligands proposed as inhibitors of this process. The ligands were evaluated for their ability to bind to and stabilize the tetrameric structure, their cooperativity in binding, and their ability to compete with the natural ligand thyroxine. The observation of a novel ten-component complex containing six protein subunits, two vitamin molecules, and two synthetic ligands allows us to conclude that ligand binding does not inhibit association of transthyretin with holo retinol binding protein.

A tandem mass spectrometer for improved transmission and analysis of large macromolecular assemblies.

We report the design and first applications of a tandem mass spectrometer (a quadrupole time-of-flight mass spectrometer) optimized for the transmission and analysis of large macromolecular assemblies. Careful control of the pressure gradient in the different pumping stages of the instrument has been found to be essential for the detection of macromolecular particles. Such assemblies are, however, difficult to analyze by tandem-MS approaches, because they give rise to signals above m/z 3,000-4,000, the limit for commercial quadrupoles. By reducing the frequency of the quadrupole to 300 kHz and using it as a narrow-band mass filter, we show that it is possible to isolate ions from a single peak at m/z 22,000 in a window as narrow as 22 m/z units. Using cesium iodide cluster signals, we show that the mass range in the time-of-flight (TOF) analyzer extends beyond m/z 90,000, in theory to more than m/z 150,000. We also demonstrate that the resolution of the instrument is greater than 3,000 at m/z 44,500. Tandem-MS capabilities are illustrated by separating components from heterooligomeric assemblies formed between tetrameric transthyretin, thyroxine, retinol-binding protein, and retinol. Isolation of a single charge state at m/z 5,340 in the quadrupole and subsequent collision-induced dissociation (CID) in the gas-filled collision cell leads to the formation of ions from individual subunits and subcomplexes, identified by their mass and charge in the TOF analyzer.

VIII International Symposium on Amyloidosis, August 7-11,1998, Rochester, MN

The VIII International Symposium on Amyloidosis was held in Rochester, Minnesota, August 7 through 11, 1998. The program consisted of six sessions, each of which was devoted to a specific area of amyloidosis research. Attendance approached 250, representing 13 countries. The session on Saturday morning opened with a talk (A. Cohen, Boston) stressing the scientific importance of and the widespread acceptance of the Society's journal AMYLOID. despite its limited subscription rate. The session was then devoted to fibril formation, P component and common elements. Dr. J. Kelly (La Jolla, California) gave an excellent address on the factors responsible for the formation of fibrils from an intermediate prefibril. He emphasized studies that demonstrated that flufenamic acid and possibly fenoprofen could be potential inhibitors of fibrillogenesis in transthyretin amyloidosis. Dr. M. Pepys (London) gave an important overview of amyloid P component and its universal association with the amyloid fibril. He noted that a knockout mouse model that lacked P component still was capable of developing amyloid. Active research is underway in anti-amyloidosis compounds that directly prevent the binding of P component to the amyloid fibril in an effort to prevent fibrillogenesis. It was also emphasized that SAP I123 scanning remains an important method of quantitating the total body burden of amyloid, as well as its ability to monitor therapy. Dr. D. Kirschner (Boston College) presented evidence suggesting that tetrameric transthyretin dissociates to a monomer as an intermediate before the formation of amyloid. The group at Indiana University reported that residues 6, 7 and 8 from serum amyloid A protein are critical for binding of HDL cholesterol. Dr. Kisilevsky (Ontario, Canada) reported that apo SAA from mouse and human contains binding sites for heparin that become available only when apo SAA dissociates from HDL. The morning presentations were completed by another presentation from Queens University, looking at early attempts to inhibit the interaction between the amyloid fibril and glycosaminoglycans as a potential site for inhibition of fibrillogenesis. There was a poster session the first morning devoted to P component and fibrillogenesis.

A highly conservative Ile-20 substitution at the dimer-dimer interface selectively reduces stability of the tetrameric transthyretin complex and is associated with familial cardiomyopathy

A highly conservative Ile-20 substitution at the dimer-dimer interface selectively reduces stability of the tetrameric transthyretin complex and is associated with familial cardiomyopathy

Crystal structure of the transthyretin--retinoic-acid complex.

Retinoids are quite insoluble and chemically unstable compounds in the aqueous medium, such that natural retinoids need to be bound to specific retinoid-binding proteins to be protected, solubilized and transported in body fluids. All-trans retinoic acid exhibits a relatively high affinity for thyroxine-binding transthyretin in vitro and this protein is a good candidate for the transport of retinoic acid administered as pharmacological or antitumor agent. To define structural features essential for the recognition by transthyretin of a ligand which is structurally unrelated to thyroxine, we have cocrystallized human transthyretin with retinoic acid and determined its structure at 0.18-nm resolution. The retinoid fits into the two chemically identical thyroxine-binding sites, which are located in the central channel that runs through the tetrameric transthyretin. The cyclohexene ring of the bound retinoid is innermost, occupying the same position of the phenolic ring of the bound 3,3'-diiodo-L-thyronine, whereas the carboxylate group, like the same group of the thyroid hormone, participates in an ionic interaction with the Lys15 side chain at the entrance of the channel. Despite the fact that transthyretin was cocrystallized with all-trans-retinoic acid, the isoprene chain of the bound retinoid has been found in a non-extended conformation. This feature, that allows the carboxylate to orient in a manner suitable for ion-pair association with the Lys15 side chain, is attributable to the conversion of all-trans-retinoic acid into cis-isomers or folded conformers. It is concluded that the presence, in an essentially hydrophobic molecular core of the appropriate size, of a negatively charged group at the correct position is a crucial requirement for ligand-transthyretin recognition. Whereas the binding of the ligand has no remarkable consequences for the protein structure, all-trans-retinoic acid undergoes structural changes such as to interact favorably with residues present in the thyroxine-binding sites, resembling roughly the natural ligand.