Beyond genetic factors in familial amyloidotic polyneuropathy: protein glycation and the loss of fibrinogen's chaperone activity

Familial amyloidotic polyneuropathy (FAP) is a systemic conformational disease characterized by extracellular amyloid fibril formation from plasma transthyretin (TTR). This is a crippling, fatal disease for which liver transplantation is the only effective therapy. More than 80 TTR point mutations are associated with amyloidotic diseases and the most widely accepted disease model relates TTR tetramer instability with TTR point mutations. However, this model fails to explain two observations. First, native TTR also forms amyloid in systemic senile amyloidosis, a geriatric disease. Second, age at disease onset varies by decades for patients bearing the same mutation and some mutation carrier individuals are asymptomatic throughout their lives. Hence, mutations only accelerate the process and non-genetic factors must play a key role in the molecular mechanisms of disease. One of these factors is protein glycation, previously associated with conformational diseases like Alzheimer's and Parkinson's. The glycation hypothesis in FAP is supported by our previous discovery of methylglyoxal-derived glycation of amyloid fibrils in FAP patients. Here we show that plasma proteins are differentially glycated by methylglyoxal in FAP patients and that fibrinogen is the main glycation target. Moreover, we also found that fibrinogen interacts with TTR in plasma. Fibrinogen has chaperone activity which is compromised upon glycation by methylglyoxal. Hence, we propose that methylglyoxal glycation hampers the chaperone activity of fibrinogen, rendering TTR more prone to aggregation, amyloid formation and ultimately, disease.     

Only amyloidogenic intermediates of transthyretin induce apoptosis

In diseases like Alzheimer's disease and familial amyloidotic polyneuropathy (FAP) amyloid deposits co-localize with areas of neurodegeneration. FAP is associated with mutations of the plasma protein transthyretin (TTR). We can here show an apoptotic effect of amyloidogenic mutants of TTR on a human neuroblastoma cell line. Toxicity could be blocked by catalase indicating a free oxygen radical dependent mechanism. The toxic effect was dependent on the state of aggregation and unexpectedly mature fibrils from FAP-patients who failed to exert an apoptotic response. Morphological studies revealed a correlation between toxicity and the presence of immature amyloid. Thus, we can show that toxicity is associated with early stages of fibril formation and propose that mature full-length fibrils represent an inert end stage, which might serve as a rescue mechanism.     

Epigallocatechin-3-gallate as a potential therapeutic drug for TTR-related amyloidosis: "in vivo" evidence from FAP mice models

Background

Familial amyloidotic polyneuropathy (FAP) is a neurodegenerative disease caused by the extracellular deposition of mutant transthyretin (TTR), with special involvement of the peripheral nervous system (PNS). Currently, hepatic transplantation is considered the most efficient therapy to halt the progression of clinical symptoms in FAP since more than 95% of TTR is produced by the liver. However, less invasive and more reliable therapeutic approaches have been proposed for FAP therapy, namely based on drugs acting as inhibitors of amyloid formation or as amyloid disruptors. We have recently reported that epigallocatechin-3-gallate (EGCG), the most abundant catechin in green tea, is able to inhibit TTR aggregation and fibril formation, “in vitro” and in a cellular system, and is also able to disrupt pre-formed amyloid fibrils “in vitro”.

Methodology and Principal Findings

In the present study, we assessed the effect of EGCG subchronic administration on TTR amyloidogenesis “in vivo”, using well characterized animal models for FAP. Semiquantitative immunohistochemistry (SQ-IHC) and Western blot analysis of mice tissues after treatment demonstrated that EGCG inhibits TTR toxic aggregates deposition in about 50% along the gastrointestinal tract (GI) and peripheral nervous system (PNS). Moreover EGCG treatment considerably lowered levels of several biomarkers associated with non-fibrillar TTR deposition, namely endoplasmic reticulum (ER)-stress, protein oxidation and apoptosis markers. Treatment of old FAP mice with EGCG resulted not only in the decrease of non-fibrillar TTR deposition but also in disaggregation of amyloid deposits. Consistently, matrix metalloproteinase (MMP)-9 and serum amyloid P component (SAP), both markers of amyloid deposition, were also found reduced in treated old FAP mice.

Conclusions and Significance

The dual effect of EGCG both as TTR aggregation inhibitor and amyloid fibril disruptor together with the high tolerability and low toxicity of EGCG in humans, point towards the potential use of this compound, or optimized derivatives, in the treatment of TTR-related amyloidoses.     

Clusterin regulates transthyretin amyloidosis

Transthyretin (TTR) is a human disease-associated amyloidogenic protein that has been implicated in senile systemic amyloidosis (SSA) and familial amyloidotic polyneuropathy (FAP). FAP typically results in severe and early-onset disease, and the only therapy established so far is liver transplantation; thus, developing new strategies for treating FAP is of paramount interest. Clusterin has recently been proposed to play a role as an extracellular molecular chaperone, affecting the fibril formation of amyloidogenic proteins. The ability of clusterin to influence amyloid fibril formation prompted us to investigate whether clusterin is capable of inhibiting TTR amyloidosis. Here, we report that clusterin strongly interacts with wild-type TTR and TTR variants V30M and L55P under acidic conditions, and blocks the amyloid fibril formation of TTR variants. In particular, the amyloid fibril formation of V30M TTR in the presence of clusterin is reduced to level similar to wild-type TTR. We also demonstrated that clusterin is an effective inhibitor of L55P TTR amyloidosis, the most aggressive form of TTR diseases. The mechanism by which clusterin inhibits TTR amyloidosis appears to be through stabilization of TTR tetrameric structure. These findings suggest the possibility of using clusterin as a therapeutic agent for TTR amyloidosis.     

Presence of N‐glycosylated transthyretin in plasma of V30M carriers in familial amyloidotic polyneuropathy: an escape from ERAD

Familial amyloid polyneuropathy (FAP) is an autosomal dominant disease characterized by deposition of amyloid related to the presence of mutations in the transthyretin (TTR) gene. TTR is mainly synthesized in liver, choroid plexuses of brain and pancreas and secreted to plasma and cerebrospinal fluid (CSF). Although it possesses a sequon for N‐glycosylation N‐D‐S at position 98, it is not secreted as a glycoprotein. The most common FAP‐associated mutation is TTR V30M. In a screening for monoclonal antibodies developed against an amyloidogenic TTR form, we detected a distinct TTR with slower electrophoretic mobility in Western of plasma from carriers of the V30M mutation, not present in normal plasma. Mass spectrometry analyses of this slower migrating TTR (SMT) identified both wild‐type and mutant V30M; SMT was undetectable upon N‐glycosidase F treatment. Furthermore, SMT readily disappeared in the plasma of V30M ‐ FAP patients after liver transplantation and appeared in plasma of transplanted domino individuals that received a V30M liver. SMT was also detected in plasma, but not in CSF of transgenic mice for the human V30M mutation. A hepatoma cell line transduced to express human V30M did not present the SMT modification in secretion media. Glycosylated TTR was absent in fibrils extracted from human kidney V30M autopsy tissue or in TTR aggregates extracted from the intestine of human TTR transgenic mice. Studies on the metabolism of this novel, glycosylated TTR secreted from FAP liver are warranted to provide new mechanisms in protein quality control and etiopathogenesis of the disease.     

Transthyretin aggregation under partially denaturing conditions is a downhill polymerization

The deposition of fibrils and amorphous aggregates of transthyretin (TTR) in patient tissues is a hallmark of TTR amyloid disease, but the molecular details of amyloidogenesis are poorly understood. Tetramer dissociation is typically rate-limiting for TTR amyloid fibril formation, so we have used a monomeric variant of TTR (M-TTR) to study the mechanism of aggregation. Amyloid formation is often considered to be a nucleation-dependent process, where fibril growth requires the formation of an oligomeric nucleus that is the highest energy species on the pathway. According to this model, the rate of fibril formation should be accelerated by the addition of preformed aggregates or "seeds", which effectively bypasses the nucleation step. Herein, we demonstrate that M-TTR amyloidogenesis at low pH is a complex, multistep reaction whose kinetic behavior is incompatible with the expectations for a nucleation-dependent polymerization. M-TTR aggregation is not accelerated by seeding, and the dependence of the reaction timecourse is first-order on the M-TTR concentration, consistent either with a dimeric nucleus or with a nonnucleated process where each step is bimolecular and essentially irreversible. These studies suggest that amyloid formation by M-TTR under partially denaturing conditions is a downhill polymerization, in which the highest energy species is the native monomer. Our results emphasize the importance of therapeutic strategies that stabilize the TTR tetramer and may help to explain why more than eighty TTR variants are disease-associated. The differences between amyloid formation by M-TTR and other amyloidogenic peptides (such as amyloid beta-peptide and islet amyloid polypeptide) demonstrate that these polypeptides do not share a common aggregation mechanism, at least under the conditions examined thus far.     

Review: TTR Amyloidosis—Structural Features Leading to Protein Aggregation and Their Implications on Therapeutic Strategies

Transthyretin amyloidosis represents a spectrum of clinical syndromes that, in all cases except senile systemic amyloidosis, are dependent on the mutation present in the transthyretin (TTR) protein. Although the role of amyloid deposits in the pathogenesis of the disease is not clear, preventing their formation or promoting their disaggregation is necessary to control the development of clinical symptoms. The design of therapies aiming at preventing amyloid formation or promoting its dissociation requires detailed knowledge of the fibrils' molecular structure and a complete view about the factors responsible for protein aggregation. This review is focused on the structural studies, performed on amyloid fibrils and amyloidogenic TTR variants, aiming at understanding the aggregation mechanism as well as the atomic structure of the fibril assembly. Based on the available information possible therapies are also surveyed.     

Review: TTR amyloidosis-structural features leading to protein aggregation and their implications on therapeutic strategies

Transthyretin amyloidosis represents a spectrum of clinical syndromes that, in all cases except senile systemic amyloidosis, are dependent on the mutation present in the transthyretin (TTR) protein. Although the role of amyloid deposits in the pathogenesis of the disease is not clear, preventing their formation or promoting their disaggregation is necessary to control the development of clinical symptoms. The design of therapies aiming at preventing amyloid formation or promoting its dissociation requires detailed knowledge of the fibrils' molecular structure and a complete view about the factors responsible for protein aggregation. This review is focused on the structural studies, performed on amyloid fibrils and amyloidogenic TTR variants, aiming at understanding the aggregation mechanism as well as the atomic structure of the fibril assembly. Based on the available information possible therapies are also surveyed.     

Cardiac amyloid in patients with familial amyloid polyneuropathy consists of abundant wild-type transthyretin

Patients with familial amyloid polyneuropathy (FAP) are now cured by liver transplantation, but cardiac amyloidosis would further progress even after liver transplantation in some patients. To clarify the pathological mechanism of the progress of cardiac amyloidosis in FAP, we investigated cardiac tissues obtained from 6 FAP patients with 3 different types of TTR mutations. One of them had undergone liver transplantation and one year later died of cardiac amyloidosis. We determined clinical severity of cardiac involvement of those patients and characterized amyloid fibril proteins depositing in their cardiac muscles by immunohistochemistry, mass spectrometry and isoelectric focusing. All the patients had cardiac dysfunction and increased cardiac weight. Diffuse deposition of TTR-related amyloid was seen in their myocardium on microscopic examination. Amyloid fibrils of the heart were composed of wild-type TTR as well as variant TTR at a ratio of about 1:1 in 5 patients without liver transplantation. In the patient with a transplanted liver, about 80% of the cardiac amyloid consisted of wild-type TTR. Wild-type TTR contributes greatly to the development of amyloid deposition in the heart of FAP patients regardless of the types of TTR mutations.     

Native state hydrogen exchange study of suppressor and pathogenic variants of transthyretin

Transthyretin (TTR) is an amyloidogenic protein whose aggregation is responsible for numerous familial amyloid diseases, the exact phenotype being dependent on the sequence deposited. Many familial disease variants display decreased stability in vitro, and early onset pathology in vivo. Only subtle structural differences were observed upon crystallographic comparison of the disease-associated variants to the T119M interallelic trans-suppressor. Herein three human TTR single amino acid variant homotetramers including two familial amyloidotic polyneuropathy (FAP) causing variants (V30M and L55P), and a suppressor variant T119M (known to protect V30M carriers from disease by trans-suppression) were investigated in a residue-specific fashion by monitoring (2)H-(1)H exchange employing NMR spectroscopy. The measured protection factors for slowly exchanging amide hydrogen atoms reveal destabilization of the protein core in the FAP variants, the core consisting of strands A, B, E and G and the loop between strands A and B. The same core exhibits much slower exchange in the suppressor variant. Accelerated exchange rates were observed for residues at the subunit interfaces in L55P, but not in the T119M or V30M TTR. The correlation between destabilization of the TTR core strands and the tendency for amyloid formation supports the view that these strands are involved in amyloidogenicity, consistent with previous (2)H-(1)H exchange analysis of the WT-TTR amyloidogenic intermediate.     

Tetramer dissociation and monomer partial unfolding precedes protofibril formation in amyloidogenic transthyretin variants

Amyloid fibril formation and deposition is a common feature of a wide range of fatal diseases including spongiform encephalopathies, Alzheimer's disease, and familial amyloidotic polyneuropathies (FAP), among many others. In certain forms of FAP, the amyloid fibrils are mostly constituted by variants of transthyretin (TTR), a homotetrameric plasma protein. Recently, we showed that transthyretin in solution may undergo dissociation to a non-native monomer, even under close to physiological conditions of temperature, pH, ionic strength, and protein concentration. We also showed that this non-native monomer is a compact structure, does not behave as a molten globule, and may lead to the formation of partially unfolded monomeric species and high molecular mass soluble aggregates (Quintas, A., Saraiva, M. J. M., and Brito, R. M. M. (1999) J. Biol. Chem. 274, 32943-32949). Here, based on aging experiments of tetrameric TTR and chemically induced protein unfolding experiments of the non-native monomeric forms, we show that tetramer dissociation and partial unfolding of the monomer precedes amyloid fibril formation. We also show that TTR variants with the least thermodynamically stable non-native monomer produce the largest amount of partially unfolded monomeric species and soluble aggregates under conditions that are close to physiological. Additionally, the soluble aggregates formed by the amyloidogenic TTR variants showed morphological and thioflavin-T fluorescence properties characteristic of amyloid. These results allowed us to conclude that amyloid fibril formation by some TTR variants might be triggered by tetramer dissociation to a compact non-native monomer with low conformational stability, which originates partially unfolded monomeric species with a high tendency for ordered aggregation into amyloid fibrils. Thus, partial unfolding and conformational fluctuations of molecular species with marginal thermodynamic stability may play a crucial role on amyloid formation in vivo.     

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 chemical chaperoning by flavonoids: Structure–activity insights towards the design of potent amyloidosis inhibitors

BackgroundMany polyphenols have been proposed as broad-spectrum inhibitors of amyloid formation. To investigate structure–activity relationships relevant for the interaction of flavonoids with transthyretin (TTR), the protein associated with familial amyloid polyneuropathy (FAP), we compared the effects of major tea catechins and their larger polymers theaflavins, side-by-side, on TTR amyloid formation process.MethodsInteraction of flavonoids with TTR and effect on TTR stability were assessed through binding assays and isoelectric focusing in polyacrylamide gel. TTR aggregation was studied, in vitro, by dynamic light scattering (DLS), transmission electron microscopy (TEM) and in cell culture, through cytotoxicity assays.ResultsTested flavonoids bound to TTR and stabilized the TTR tetramer, with different potencies. The flavonoids also inhibited in vitro formation of TTR small oligomeric species and in cell culture inhibited pathways involving caspase-3 activation and ER stress that are induced by TTR oligomers. In all assays performed the galloyl esters presented higher potency to inhibit aggregation than the non-gallated flavonoids tested.ConclusionsOur results highlight the presence of gallate ester moiety as key structural feature of flavonoids in chemical chaperoning of TTR aggregation. Upon binding to the native tetramer, gallated flavonoids redirect the TTR amyloidogenic pathway into unstructured nontoxic aggregation assemblies more efficiently than their non-gallated forms.General significanceOur findings suggest that galloyl moieties greatly enhance flavonoid anti-amyloid chaperone activity and this should be taken into consideration in therapeutic candidate drug discovery.     

Systemic amyloidosis in transgenic mice carrying the human mutant transthyretin (Met 30) gene

To analyze the pathologic processes of amyloid deposition in type I familial amyloidotic polyneuropathy (FAP), mice were made transgenic by introducing the human mutant transthyretin (TTR) gene(MT-hMet 30). An inbred strain of mouse, C57 BL/6, was chosen. Transgenic mice were killed using ether anesthesia at 3-mo intervals up to 24 mo after birth. In these transgenic mice, amyloid deposition started in the gastrointestinal tract, cardiovascular system, and kidneys and extended to various other organs and tissues with advancing age. The pattern of amyloid deposition was similar to that observed in human autopsy cases of FAP, except for its absence in the choroid plexus and in the peripheral and autonomic nervous systems. We extracted the amyloid fibrils from kidneys of these mice with a human mutant TTR gene and analyzed them immunochemically and electronmicroscopically. Deposited amyloid was shown to be composed of human mutant TTR and mouse serum amyloid P component. Amyloid fibril from transgenic mice was morphologically and immunohistochemically similar to that of human FAP. The most striking pathologic feature of the transgenic mice was the absence of amyloid deposition in the peripheral and autonomic nervous tissues. Thus, other intrinsic factors may be involved in amyloid deposition in the nervous tissues of human FAP.     

Cholesterol and anionic phospholipids increase the binding of amyloidogenic transthyretin to lipid membranes

Deposition of transthyretin (TTR) amyloid is a pathological hallmark of familial amyloidotic polyneuropathy (FAP). Recently we showed that TTR binds to membrane lipids via electrostatic interactions and that membrane binding is correlated with the cytotoxicity induced by amyloidogenic TTR. In the present study, we examined the role of lipid composition in membrane binding of TTR by a surface plasmon resonance (SPR) approach. TTR bound to lipid bilayers through both high- and low-affinity interactions. Increasing the mole fraction of cholesterol in the bilayer led to an increase in the amount of high-affinity binding of an amyloidogenic mutant (L55P) TTR. In addition, a greater amount of L55P TTR bound with high affinity to membranes made from anionic phospholipids, phosphatidylglycerol (PG) and phosphatidylserine (PS), than to membranes made from zwitterionic phospholipid phosphatidylcholine (PC). The anionic phospholipids (PS and PG) promoted the aggregation of L55P TTR by accelerating the nucleation phase of aggregation, whereas the zwitterionic phospholipid PC had little effect. These results suggest that cholesterol and anionic phospholipids may be important for TTR aggregation and TTR-induced cytotoxicity.     

Cholesterol and anionic phospholipids increase the binding of amyloidogenic transthyretin to lipid membranes

Deposition of transthyretin (TTR) amyloid is a pathological hallmark of familial amyloidotic polyneuropathy (FAP). Recently we showed that TTR binds to membrane lipids via electrostatic interactions and that membrane binding is correlated with the cytotoxicity induced by amyloidogenic TTR. In the present study, we examined the role of lipid composition in membrane binding of TTR by a surface plasmon resonance (SPR) approach. TTR bound to lipid bilayers through both high- and low-affinity interactions. Increasing the mole fraction of cholesterol in the bilayer led to an increase in the amount of high-affinity binding of an amyloidogenic mutant (L55P) TTR. In addition, a greater amount of L55P TTR bound with high affinity to membranes made from anionic phospholipids, phosphatidylglycerol (PG) and phosphatidylserine (PS), than to membranes made from zwitterionic phospholipid phosphatidylcholine (PC). The anionic phospholipids (PS and PG) promoted the aggregation of L55P TTR by accelerating the nucleation phase of aggregation, whereas the zwitterionic phospholipid PC had little effect. These results suggest that cholesterol and anionic phospholipids may be important for TTR aggregation and TTR-induced cytotoxicity.     

Anti-apoptotic treatment reduces transthyretin deposition in a transgenic mouse model of Familial Amyloidotic Polyneuropathy

Tauroursodeoxycholic acid (TUDCA) is a unique natural compound that acts as a potent anti-apoptotic and anti-oxidant agent, reducing cytotoxicity in several neurodegenerative diseases. Since oxidative stress, apoptosis and inflammation are associated with transthyretin (TTR) deposition in Familial Amyloidotic Polyneuropathy (FAP), we investigated the possible TUDCA therapeutical application in this disease. We show by semi-quantitative immunohistochemistry and western blotting that administration of TUDCA to a transgenic mouse model of FAP decreased apoptotic and oxidative biomarkers usually associated with TTR deposition, namely the ER stress markers BiP and eIF2alpha, the Fas death receptor and oxidation products such as 3-nitrotyrosine. Most important, TUDCA treatment significantly reduced TTR toxic aggregates in as much as 75%. Since TUDCA has no effect on TTR aggregation "in vitro", this finding points for the "in vivo" modulation of TTR aggregation by cellular responses, such as by oxidative stress, ER stress and apoptosis and prompts for the use of this safe drug in prophylactic and therapeutic measures in FAP.     

Transthyretin amyloidosis: a tale of weak interactions.

Over 70 transthyretin (TTR) mutations have been associated with hereditary amyloidoses, which are all autosomal dominant disorders with adult age of onset. TTR is the main constituent of amyloid that deposits preferentially in peripheral nerve giving rise to familial amyloid polyneuropathy (FAP), or in the heart leading to familial amyloid cardiomyopathy. Since the beginning of this decade the central question of these types of amyloidoses has been why TTR is an amyloidogenic protein with clinically heterogeneous pathogenic consequences. As a result of amino acid substitutions, conformational changes occur in the molecule, leading to weaker subunit interactions of the tetrameric structure as revealed by X-ray studies of some amyloidogenic mutants. Modified soluble tetramers exposing cryptic epitopes seem to circulate in FAP patients as evidenced by antibody probes recognizing specifically TTR amyloid fibrils, but what triggers dissociation into monomeric and oligomeric intermediates of amyloid fibrils is largely unknown. Avoiding tetramer dissociation and disrupting amyloid fibrils are possible avenues of therapeutic intervention based on current molecular knowledge of TTR amyloidogenesis and fibril structure.     

Trapping the monomer of a non-amyloidogenic variant of transthyretin: exploring its possible use as a therapeutic strategy against transthyretin amyloidogenic diseases

Transthyretin (TTR) is a 127-residue homotetrameric beta-sheet-rich protein that transports thyroxine in the blood and cerebrospinal fluid. The deposition of fibrils and amorphous aggregates of TTR in patients' tissues is a hallmark of TTR amyloid disease. Familial amyloidotic polyneuropathy is a hereditary form of TTR amyloidosis that is associated with one among 80 different variants of TTR. The most aggressive variants of TTR are V30M, L55P, and A25T, and the propensity to undergo aggregation seems to be linked to tetramer stability. T119M is a very stable, non-amyloidogenic variant of TTR. Here we show that the combination of high hydrostatic pressure with subdenaturing concentrations of urea (4 m) at 1 degrees C irreversibly dissociates T119M into monomers in less than 30 min in a concentration-dependent fashion. After pressure and urea removal, long lived monomers are the only species present in solution. We took advantage of the slow reassociation kinetics of these monomers into tetramers to produce heterotetramers by mixing the T119M monomers with the tetramers of the aggressive mutants of TTR. Our data show that T119M monomers can be successfully incorporated into all of these tetramers even when the exchange is performed in a more physiological environment such as human plasma; these monomers render the resultant heterotetramers less amyloidogenic. The data presented here are relevant for the understanding of T119M folding and association reactions and provide a protocol for producing T119M monomers that function as inhibitors of TTR aggregation when incorporated in to tetramers. This protocol may provide a new strategy for treating TTR diseases for which there is no therapy available other than liver transplantation.     

Structure and assembly-disassembly properties of wild-type transthyretin amyloid protofibrils observed with atomic force microscopy

Transthyretin (TTR) is an important human transport protein present in the serum and the cerebrospinal fluid. Aggregation of TTR in the form of amyloid fibrils is associated with neurodegeneration, but the mechanisms of cytotoxicity are likely to stem from the presence of intermediate assembly states. Characterization of these intermediate species is therefore essential to understand the etiology and pathogenesis of TTR-related amyloidoses. In the present work we used atomic force microscopy to investigate the morphological features of wild-type (WT) TTR amyloid protofibrils that appear in the early stages of aggregation. TTR protofibrils obtained by mild acidification appeared as flexible filaments with variable length and were able to bind amyloid markers (thioflavin T and Congo red). Surface topology and contour-length distribution displayed a periodic pattern of ～ 15 nm, suggesting that the protofibrils assemble via an end-binding oligomer fusion mechanism. The average height and periodic substructure found in protofibrils is compatible with the double-helical model of the TTR amyloid protofilament. Over time protofibrils aggregated into bundles and did not form mature amyloid-like fibrils. Unlike amyloid fibrils that are typically stable under physiological conditions, the bundles dissociated into component protofibrils with axially compacted and radially dilated structure when exposed to phosphate-buffered saline solution. Thus, WT TTR can form metastable filamentous aggregates that may represent an important transient state along the pathway towards the formation of cytotoxic TTR species.