Arrangement of subunits and ordering of beta-strands in an amyloid sheet

Amyloid fibrils are associated with several disease states, but their structures have yet to be fully defined. Here we use site-directed spin labeling to explain some of the specific interactions that are formed between subunits when the protein transthyretin (TTR) assembles into amyloid fibrils, which are associated with both spontaneous and familial amyloid diseases in humans. The results suggest that fibrils are formed when a major conformational change displaces the terminal beta-strand from the edge of a beta-sheet in the native structure, exposing the penultimate strand. The newly exposed strand then allows a novel beta-sheet interaction to form between the TTR subunits. This interaction and another previously identified subunit association lead to a plausible model for the specific sequence of beta-strands in one of the indefinitely repeating beta-sheets of TTR amyloid, which is formed by a head-to-head, tail-to-tail arrangement of subunits.     

Uncovering the Mechanism of Aggregation of Human Transthyretin

The tetrameric thyroxine transport protein transthyretin (TTR) forms amyloid fibrils upon dissociation and monomer unfolding. The aggregation of transthyretin has been reported as the cause of the life-threatening transthyretin amyloidosis. The standard treatment of familial cases of TTR amyloidosis has been liver transplantation. Although aggregation-preventing strategies involving ligands are known, understanding the mechanism of TTR aggregation can lead to additional inhibition approaches. Several models of TTR amyloid fibrils have been proposed, but the segments that drive aggregation of the protein have remained unknown. Here we identify β-strands F and H as necessary for TTR aggregation. Based on the crystal structures of these segments, we designed two non-natural peptide inhibitors that block aggregation. This work provides the first characterization of peptide inhibitors for TTR aggregation, establishing a novel therapeutic strategy.     

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.     

The most pathogenic transthyretin variant, L55P, forms amyloid fibrils under acidic conditions and protofilaments under physiological conditions.

The L55P transthyretin (TTR) familial amyloid polyneuropathy-associated variant is distinct from the other TTR variants studied to date and the wild-type protein in that the L55P tetramer can dissociate to the monomeric amyloidogenic intermediate and form fibril precursors under physiological conditions (pH 7.0, 37 degrees C). The activation barrier associated with L55P-TTR tetramer dissociation is lower than the barrier for wild-type transthyretin dissociation, which does not form fibrils under physiological conditions. The L55P-TTR tetramer is also very sensitive to acidic conditions, readily dissociating to form the monomeric amyloidogenic intermediate between pH 5.5-5.0 where the wild-type TTR adopts a nonamyloidogenic tetrameric structure. The formation of the L55P monomeric amyloidogenic intermediate involves subtle tertiary structural changes within the beta-sheet rich subunit as discerned from Trp fluorescence, circular dichroism analysis, and ANS binding studies. The assembly of the L55P-TTR amyloidogenic intermediate at physiological pH (pH 7.5) affords protofilaments that elongate with time. TEM studies suggest that the entropic barrier associated with filament assembly (amyloid fibril formation) is high in vitro, amyloid being defined by the laterally assembled four filament structure observed by Blake upon isolation of "fibrils" from the eye of a FAP patient. The L55P-TTR protofilaments formed in vitro bind Congo red and thioflavin T (albeit more weakly than the fibrils produced at acidic pH), suggesting that the structure observed probably represents an amyloid precursor. The structural continuum from misfolded monomer through protofilaments, filaments, and ultimately fibrils must be considered as a possible source of pathology associated with these diseases.     

Natural polyphenols inhibit different steps of the process of transthyretin (TTR) amyloid fibril formation

Several natural polyphenols with potent inhibitory effects on amyloid fibril formation have been reported. Herein, we studied modulation of transthyretin (TTR) fibrillogenesis by selected polyphenols. We demonstrate that both curcumin and nordihydroguaiaretic acid (NDGA) bind to TTR and stabilize the TTR tetramer. However, while NDGA slightly reduced TTR aggregation, curcumin strongly suppressed TTR amyloid fibril formation by generating small "off-pathway" oligomers and EGCG maintained most of the protein in a non-aggregated soluble form. This indicates alternative mechanisms of action supported by the occurrence of different non-toxic intermediates. Moreover, EGCG and curcumin efficiently disaggregated pre-formed TTR amyloid fibrils. Our studies, together with the safe toxicological profile of these phytochemicals may guide a novel pharmacotherapy for TTR-related amyloidosis targeting different steps in fibrillogenesis.     

Dissecting the structure, thermodynamic stability, and aggregation properties of the A25T transthyretin (A25T-TTR) variant involved in leptomeningeal amyloidosis: identifying protein partners that co-aggregate during A25T-TTR fibrillogenesis in cerebrospinal fluid

Deposition of amorphous aggregates and fibrils of transthyretin (TTR) in leptomeninges and subarachnoid vessels is a characteristic of leptomeningeal amyloidosis (LA), a currently untreatable cerebral angiopathy. Herein, we report the X-ray structure of the A25T homotetramer of TTR, a natural mutant described in a patient with LA. The structure of A25T-TTR is indistinguishable from that of wild-type TTR (wt-TTR), indicating that the difference in amyloidogenicity between A25T-TTR and wt-TTR cannot be ascribed to gross structural differences. Using pressure-induced dissociation of the tetramer, we show that A25T-TTR is 3 kcal/mol less stable than L55P-TTR, the most aggressive mutant of TTR described to date. After incubation for 15 days at 37 °C (pH 7.3), A25T-TTR forms mature amyloid fibrils. To mimic the environment in which TTR aggregates, we investigated aggregation in cerebrospinal fluid (CSF). Unlike L55P-TTR, A25T-TTR rapidly forms amyloid aggregates in CSF that incorporated several protein partners. Utilizing a proteomics methodology, we identified 19 proteins that copurified with A25T-TTR amyloid fibrils. We confirmed the presence of proteins previously identified to be associated with TTR aggregates in biopsies of TTR amyloidosis patients, such as clusterin, apolipoprotein E, and complement proteins. Moreover, we identified novel proteins, such as blood coagulation proteins. Overall, our results revealed the in vitro characterization of TTR aggregation in a biologically relevant environment, opening new avenues of investigation into the molecular mechanisms of LA.     

Partial denaturation of transthyretin is sufficient for amyloid fibril formation in vitro.

Amyloid diseases are caused by the self-assembly of a given protein into an insoluble cross-beta-sheet quaternary structural form which is pathogenic. An understanding of the biochemical mechanism of amyloid fibril formation should prove useful in understanding amyloid disease. Toward this end, a procedure for the conversion of the amyloidogenic protein transthyretin into amyloid fibrils under conditions which mimic the acidic environment of a lysosome has been developed. Association of a structured transthyretin denaturation intermediate is sufficient for amyloid fibril formation in vitro. The rate of fibril formation is pH dependent with significant rates being observed at pHs accessible within the lysosome (3.6-4.8). Far-UV CD spectroscopic studies suggest that transthyretin retains its secondary structural features at pHs where fibrils are formed. Near-UV CD studies demonstrate that transthyretin has retained the majority of its tertiary structure during fibril formation as well. Near-UV CD analysis in combination with glutaraldehyde cross-linking studies suggests that a pH-mediated tetramer to monomer transition is operative in the pH range where fibril formation occurs. The rate of fibril formation decreases markedly at pHs below pH 3.6, consistent with denaturation to a monomeric TTR intermediate which has lost its native tertiary structure and capability to form fibrils. It is difficult to specify with certainty which quaternary structural form of transthyretin is the amyloidogenic intermediate at this time. These difficulties arise because the maximal rate of fibril formation occurs at pH 3.6 where tetramer, traces of dimer, and significant amounts of monomer are observed.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

A comparative analysis of 23 structures of the amyloidogenic protein transthyretin

Self-assembly of the human plasma protein transthyretin (TTR) into unbranched insoluble amyloid fibrils occurs as a result of point mutations that destabilize the molecule, leading to conformational changes. The tertiary structure of native soluble TTR and many of its disease-causing mutants have been determined. Several independent studies by X-ray crystallography have suggested structural differences between TTR variants which are claimed to be of significance for amyloid formation. As these changes are minor and not consistent between the studies, we have compared all TTR structures available at the protein data bank including three wild-types, three non-amyloidogenic mutants, seven amyloidogenic mutants and nine complexes. The reference for this study is a new 1.5 A resolution structure of human wild-type TTR refined to an R-factor/R-free of 18.6 %/21.6 %. The present findings are discussed in the light of the previous structural studies of TTR variants, and show the reported structural differences to be non-significant.     

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 48 h of incubation. More mature fibrils (>24 h 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.     

Force spectroscopy reveals the presence of structurally modified dimers in transthyretin amyloid annular oligomers

Toxicity in amyloidogenic protein misfolding disorders is thought to involve intermediate states of aggregation associated with the formation of amyloid fibrils. Despite their relevance, the heterogeneity and transience of these oligomers have placed great barriers in our understanding of their structural properties. Among amyloid intermediates, annular oligomers or annular protofibrils have raised considerable interest because they may contribute to a mechanism of cellular toxicity via membrane permeation. Here we investigated, by using AFM force spectroscopy, the structural detail of amyloid annular oligomers from transthyretin (TTR), a protein involved in systemic and neurodegenerative amyloidogenic disorders. Manipulation was performed in situ , in the absence of molecular handles and using persistence length‐fit values to select relevant curves. Force curves reveal the presence of dimers in TTR annular oligomers that unfold via a series of structural intermediates. This is in contrast with the manipulation of native TTR that was more often manipulated over length scales compatible with a TTR monomer and without unfolding intermediates. Imaging and force spectroscopy data suggest that dimers are formed by the assembly of monomers in a head‐to‐head orientation with a nonnative interface along their β‐strands. Furthermore, these dimers stack through nonnative contacts that may enhance the stability of the misfolded structure.     

Stacked Sets of Parallel, In-register ��-Strands of ��2-Microglobulin in Amyloid Fibrils Revealed by Site-directed Spin Labeling and Chemical Labeling

β₂-microglobulin (β₂m) is a 99-residue protein with an immunoglobulin fold that forms β-sheet-rich amyloid fibrils in dialysis-related amyloidosis. Here the environment and accessibility of side chains within amyloid fibrils formed in vitro from β₂m with a long straight morphology are probed by site-directed spin labeling and accessibility to modification with N-ethyl maleimide using 19 site-specific cysteine variants. Continuous wave electron paramagnetic resonance spectroscopy of these fibrils reveals a core predominantly organized in a parallel, in-register arrangement, by contrast with other β₂m aggregates. A continuous array of parallel, in-register β-strands involving most of the polypeptide sequence is inconsistent with the cryoelectron microscopy structure, which reveals an architecture based on subunit repeats. To reconcile these data, the number of spins in close proximity required to give rise to spin exchange was determined. Systematic studies of a model protein system indicated that juxtaposition of four spin labels is sufficient to generate exchange narrowing. Combined with information about side-chain mobility and accessibility, we propose that the amyloid fibrils of β₂m consist of about six β₂m monomers organized in stacks with a parallel, in-register array. The results suggest an organization more complex than the accordion-like β-sandwich structure commonly proposed for amyloid fibrils.     

Disease-associated mutations impacting BC-loop flexibility trigger long-range transthyretin tetramer destabilization and aggregation

Hereditary transthyretin amyloidosis (ATTR) is an autosomal dominant disease characterized by the extracellular deposition of the transport protein transthyretin (TTR) as amyloid fibrils. Despite the progress achieved in recent years, understanding why different TTR residue substitutions lead to different clinical manifestations remains elusive. Here, we studied the molecular basis of disease-causing missense mutations affecting residues R34 and K35. R34G and K35T variants cause vitreous amyloidosis, whereas R34T and K35N mutations result in amyloid polyneuropathy and restrictive cardiomyopathy. All variants are more sensitive to pH-induced dissociation and amyloid formation than the wild-type (WT)-TTR counterpart, specifically in the variants deposited in the eyes amyloid formation occurs close to physiological pHs. Chemical denaturation experiments indicate that all the mutants are less stable than WT-TTR, with the vitreous amyloidosis variants, R34G and K35T, being highly destabilized. Sequence-induced stabilization of the dimer–dimer interface with T119M rendered tetramers containing R34G or K35T mutations resistant to pH-induced aggregation. Because R34 and K35 are among the residues more distant to the TTR interface, their impact in this region is therefore theorized to occur at long range. The crystal structures of double mutants, R34G/T119M and K35T/T119M, together with molecular dynamics simulations indicate that their strong destabilizing effect is initiated locally at the BC loop, increasing its flexibility in a mutation-dependent manner. Overall, the present findings help us to understand the sequence-dynamic-structural mechanistic details of TTR amyloid aggregation triggered by R34 and K35 variants and to link the degree of mutation-induced conformational flexibility to protein aggregation propensity.     

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.     

In vitro inhibition of transthyretin aggregate-induced cytotoxicity by full and peptide derived forms of the soluble receptor for advanced glycation end products (RAGE)

Familial amyloidotic polyneuropathy is a neurodegenerative disorder characterized by systemic extracellular deposition of transthyretin (TTR) amyloid fibrils. The latter have been proposed to trigger neurodegeneration through engagement of the receptor for advanced glycation end products (RAGE). Here we show that TTR interaction with RAGE is conserved across mouse and human species and is not dependent on RAGE glycosylation. Moreover, strand D of TTR structure seems important for the TTR-RAGE interaction as well as a motif in RAGE (residues 102-118) located within the V-domain; this motif suppressed TTR aggregate-induced cytotoxicity in cell culture.     

Cardiac amyloidosis: the need for early diagnosis

Amyloidosis is a collection of systemic diseases characterised by misfolding of previously soluble precursor proteins that become infiltrative depositions, thereby disrupting normal organ structure and function. In the heart, accumulating amyloid fibrils lead to progressive ventricular wall thickening and stiffness, resulting in diastolic dysfunction gradually progressing to a restrictive cardiomyopathy. The main types of cardiac amyloidosis are amyloid light chain (AL) amyloidosis caused by an underlying plasma cell dyscrasia, amyloid transthyretin (TTR) amyloidosis of wild-type (normal) TTR at older age (ATTRwt) and hereditary or mutant amyloid TTR (ATTRm) in which a genetic mutation leads to an unstable TTR protein. Overall survival is poor once heart failure develops, underlining the need for early referral and diagnosis. Treatment for AL amyloidosis has improved markedly over the last decades, and TTR amyloidosis gene silencers and orally available transthyretin stabilisers are ready to enter the clinical arena after recent positive outcome trials. Novel therapies aiming at fibril degradation with monoclonal antibodies are under investigation. In this review, we focus on ‘red flag’ signs and symptoms, diagnosis and management of cardiac amyloidosis which differs considerably from the general management of heart failure. Only by increasing awareness, prognosis for patients with this devastating disease can be improved.

     

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.     

Rational design of potent human transthyretin amyloid disease inhibitors

The human amyloid disorders, familial amyloid polyneuropathy, familial amyloid cardiomyopathy and senile systemic amyloidosis, are caused by insoluble transthyretin (TTR) fibrils, which deposit in the peripheral nerves and heart tissue. Several nonsteroidal anti-inflammatory drugs and structurally similar compounds have been found to strongly inhibit the formation of TTR amyloid fibrils in vitro. These include flufenamic acid, diclofenac, flurbiprofen, and resveratrol. Crystal structures of the protein-drug complexes have been determined to allow detailed analyses of the protein-drug interactions that stabilize the native tetrameric conformation of TTR and inhibit the formation of amyloidogenic TTR. Using a structure-based drug design approach ortho-trifluormethylphenyl anthranilic acid and N-(meta-trifluoromethylphenyl) phenoxazine 4, 6-dicarboxylic acid have been discovered to be very potent and specific TTR fibril formation inhibitors. This research provides a rationale for a chemotherapeutic approach for the treatment of TTR-associated amyloid diseases.     

Bifunctional crosslinking ligands for transthyretin

Wild-type and variant forms of transthyretin (TTR), a normal plasma protein, are amyloidogenic and can be deposited in the tissues as amyloid fibrils causing acquired and hereditary systemic TTR amyloidosis, a debilitating and usually fatal disease. Reduction in the abundance of amyloid fibril precursor proteins arrests amyloid deposition and halts disease progression in all forms of amyloidosis including TTR type. Our previous demonstration that circulating serum amyloid P component (SAP) is efficiently depleted by administration of a specific small molecule ligand compound, that non-covalently crosslinks pairs of SAP molecules, suggested that TTR may be also amenable to this approach. We first confirmed that chemically crosslinked human TTR is rapidly cleared from the circulation in mice. In order to crosslink pairs of TTR molecules, promote their accelerated clearance and thus therapeutically deplete plasma TTR, we prepared a range of bivalent specific ligands for the thyroxine binding sites of TTR. Non-covalently bound human TTR–ligand complexes were formed that were stable in vitro and in vivo, but they were not cleared from the plasma of mice in vivo more rapidly than native uncomplexed TTR. Therapeutic depletion of circulating TTR will require additional mechanisms.