Full-length TTR Research Articles

Trypsin-induced aggregation of transthyretin Valine 30 variants associated with hereditary amyloidosis.

Amyloid fibrils of transthyretin (TTR) consist of full-length TTR and C-terminal fragments starting near residue 50. However, the molecular mechanism underlying the production of the C-terminal fragment remains unclear. Here, we investigated trypsin-induced aggregation and urea-induced unfolding of TTR variants associated with hereditary amyloidosis. Trypsin strongly induced aggregation of variants V30G and V30A, in each of which Val30 in the hydrophobic core of the monomer was mutated to less-bulky amino acids. Variants V30L and V30M, in each of which Val30 was mutated to bulky amino acids, also exhibited trypsin-induced aggregation. On the other hand, pathogenic variant I68L as well as the nonpathogenic V30I did not exhibit trypsin-induced aggregation. The V30G variant was extremely unstable compared with the other variants. The V30G mutation caused the formation of a cavity and the rearrangement of Leu55 in the hydrophobic core of the monomer. These results suggest that highly destabilized transthyretin variants are more susceptible to trypsin digestion.

In Vitro and In Vivo Effects of SerpinA1 on the Modulation of Transthyretin Proteolysis.

Transthyretin (TTR) proteolysis has been recognized as a complementary mechanism contributing to transthyretin-related amyloidosis (ATTR amyloidosis). Accordingly, amyloid deposits can be composed mainly of full-length TTR or contain a mixture of both cleaved and full-length TTR, particularly in the heart. The fragmentation pattern at Lys48 suggests the involvement of a serine protease, such as plasmin. The most common TTR variant, TTR V30M, is susceptible to plasmin-mediated proteolysis, and the presence of TTR fragments facilitates TTR amyloidogenesis. Recent studies revealed that the serine protease inhibitor, SerpinA1, was differentially expressed in hepatocyte-like cells (HLCs) from ATTR patients. In this work, we evaluated the effects of SerpinA1 on in vitro and in vivo modulation of TTR V30M proteolysis, aggregation, and deposition. We found that plasmin-mediated TTR proteolysis and aggregation are partially inhibited by SerpinA1. Furthermore, in vivo downregulation of SerpinA1 increased TTR levels in mice plasma and deposition in the cardiac tissue of older animals. The presence of TTR fragments was observed in the heart of young and old mice but not in other tissues following SerpinA1 knockdown. Increased proteolytic activity, particularly plasmin activity, was detected in mice plasmas. Overall, our results indicate that SerpinA1 modulates TTR proteolysis and aggregation in vitro and in vivo.

Transthyretin amyloid deposits in lumbar spinal stenosis and assessment of signs of systemic amyloidosis.

BackgroundWild‐type transthyretin (ATTRwt) amyloidosis is the most common systemic amyloidosis in Western countries and manifests mainly as progressive restrictive cardiomyopathy.ObjectiveTo study the prevalence of ATTR deposits in ligament tissue in patients undergoing surgery for lumbar spinal stenosis and to assess whether these deposits are associated with cardiac amyloidosis.Materials and methodsA total of 250 patients, aged 50–89 (57% women), none with known cardiovascular disease, were included. Ligaments were investigated microscopically for amyloid. ATTR type was determined by immunohistochemistry and fibril type by Western blot. The amount of amyloid was graded 0‐4. All patients with grade 3‐4 ATTR deposits were offered cardiac investigation including ECG, cardiac ultrasound, plasma NT‐proBNP and cardiac magnetic resonance (CMR), including modern tissue characterization.ResultsAmyloid was identified in 221 of the samples (88.4%). ATTR appeared in 93 samples (37%) of whom 42 (17 women and 25 men) were graded 3‐4; all had fibril type A (mixture of full‐length TTR and fragmented TTR). Twenty‐nine of 42 patients with grade 3‐4 ATTR deposits accepted cardiovascular investigations; none of them had definite signs of cardiac amyloidosis, but five men had a history of carpal tunnel syndrome.ConclusionsThe prevalence of ATTR deposits in ligamentum flavum in patients with lumbar spinal stenosis was high but not associated with manifest ATTR cardiac amyloidosis. However, the findings of fibril type A, the prevalence of previous carpal tunnel syndrome and ATTR amyloid in surrounding adipose and vascular tissue indicate that amyloid deposits in ligamentum flavum may be an early manifestation of systemic ATTR disease.

Disruption of the CD Loop by Enzymatic Cleavage Promotes the Formation of Toxic Transthyretin Oligomers through a Common Transthyretin Misfolding Pathway.

Amyloid formation of full-length TTR involves dissociation of the native tetramers into misfolded monomers that self-assemble into amyloid. In addition to the full-length TTR, C-terminal fragments including residues 49-127 were also observed in vivo, implying the presence of additional misfolding pathways. It was previously proposed that a proteolytic cleavage might lead to the formation of the C-terminal fragment TTR amyloid. Here, we report mechanistic studies of misfolding and aggregation of a TTR variant (G53A) in the absence and presence of a serine protease. A proteolytic cleavage of G53A in the CD loop (K48 and T49) with agitation promoted TTR misfolding and aggregation, suggesting that the proteolytic cleavage may lead to the aggregation of the C-terminal fragment (residues 49-127). To gain more detailed insights into TTR misfolding promoted by proteolytic cleavage, we investigated structural changes in G53A TTR in the presence and absence of trypsin. Our combined biophysical analyses revealed that the proteolytic cleavage accelerated the formation of spherical small oligomers, which exhibited cytotoxic activities. However, the truncated TTR appeared to maintain native-like structures, rather than the C-terminal fragment (residues 49-127) being released and unfolded from the native state. In addition, our solid-state nuclear magnetic resonance and Fourier transform infrared structural studies showed that the two aggregates derived from the full-length and cleaved TTR exhibited nearly identical molecular structural features, suggesting that the proteolytic cleavage in the CD loop destabilizes the native tetrameric structure and accelerates oligomer formation through a common TTR misfolding and aggregation mechanism rather than through a distinct molecular mechanism.

An evaluation of patisiran: a viable treatment option for transthyretin-related hereditary amyloidosis

ABSTRACTIntroduction: Hereditary transthyretin-mediated amyloidosis (ATTRv; v for variant) is a rare, progressive, fatal multi-systemic disease, autosomal dominantly inherited with heterogeneous clinical phenotype caused by mutations in the TTR gene. Mutations promoting proteolytic remodeling and tetramer dissociation result in fragmented and full-length TTR monomers that misfold, aggregate and deposit at multiple sites (mainly nerves and heart) causing peripheral neuropathy and/or cardiomyopathy.Areas covered: The authors discuss patisiran, the first approved RNA interference-based therapeutic agent that suppresses the circulating levels of the amyloidogenic protein TTR both wild-type and mutant. This compound demonstrated a safe clinical profile in phase I and II studies and showed a significant clinical effect in a phase III (APOLLO) trial in ATTRv patients. An open-label-extension study is still underway but, based on the positive results, the regulatory agencies granted approval for the treatment of ATTRv with polyneuropathy in Stage I and II.Expert opinion: The patisiran program has demonstrated that substantial TTR concentration reduction is associated with significant and sustained improvement in polyneuropathy scores, quality-of-life profile and several outcome measures that capture the systemic burden of the disease. The drug resulted safe also in long term follow-up studies while its efficacy for ATTR with cardiomyopathy is under investigation.

A cell-based high-throughput screening method to directly examine transthyretin amyloid fibril formation at neutral pH

Transthyretin (TTR) is a major amyloidogenic protein associated with hereditary (ATTRm) and nonhereditary (ATTRwt) intractable systemic transthyretin amyloidosis. The pathological mechanisms of ATTR-associated amyloid fibril formation are incompletely understood, and there is a need for identifying compounds that target ATTR. C-terminal TTR fragments are often present in amyloid-laden tissues of most patients with ATTR amyloidosis, and on the basis of in vitro studies, these fragments have been proposed to play important roles in amyloid formation. Here, we found that experimentally-formed aggregates of full-length TTR are cleaved into C-terminal fragments, which were also identified in patients' amyloid-laden tissues and in SH-SY5Y neuronal and U87MG glial cells. We observed that a 5-kDa C-terminal fragment of TTR, TTR81-127, is highly amyloidogenic in vitro, even at neutral pH. This fragment formed amyloid deposits and induced apoptosis and inflammatory gene expression also in cultured cells. Using the highly amyloidogenic TTR81-127 fragment, we developed a cell-based high-throughput screening method to discover compounds that disrupt TTR amyloid fibrils. Screening a library of 1280 off-patent drugs, we identified two candidate repositioning drugs, pyrvinium pamoate and apomorphine hydrochloride. Both drugs disrupted patient-derived TTR amyloid fibrils ex vivo, and pyrvinium pamoate also stabilized the tetrameric structure of TTR ex vivo in patient plasma. We conclude that our TTR81-127-based screening method is very useful for discovering therapeutic drugs that directly disrupt amyloid fibrils. We propose that repositioning pyrvinium pamoate and apomorphine hydrochloride as TTR amyloid-disrupting agents may enable evaluation of their clinical utility for managing ATTR amyloidosis.

Transthyretin Glu54Leu – an unknown mutation within the Swedish population associated with amyloid cardiomyopathy and a unique fibril type

For the first time, we report of a Swedish family of five individuals with a TTR Glu54Leu (p. Glu74Leu) mutation in the transthyretin gene. This mutation has been previously described a few times in the literature, but no phenotypic or clinical description has been done before. The most common mutation in the Swedish population is TTRVal30Met and is mostly found in the Northern part of Sweden. Interestingly, the TTRGlu54Leu mutation was found in the same endemic area. The main phenotype of the TTR Glu54Leu patients is severe cardiomyopathy, which resulted in heart transplantation for the index person. As previously seen for ATTR amyloidosis patients with mainly cardiomyopathy, the amyloid fibrils consisted of a mixture of full-length and fragmented TTR species. However, western blot analyses detected a previously unrecognized band, indicating that these patients may have a third, so far unrecognized, fibril composition type that is distinct from the usual type A band pattern.

Amyloid fibril composition within hereditary Val30Met (p. Val50Met) transthyretin amyloidosis families.

BackgroundThe amyloid fibril in hereditary transthyretin (TTR) Val30Met (pVal50Met) amyloid (ATTR Val30Met) amyloidosis is composed of either a mixture of full-length and TTR fragments (Type A) or of only full-length TTR (Type B). The type of amyloid fibril exerts an impact on the phenotype of the disease, and on the outcome of diagnostic procedures and therapy. The aim of the present study was to investigate if the type of amyloid fibril remains the same within ATTR Val30Met amyloidosis families.MethodsFifteen families were identified in whom at least two first-degree relatives had their amyloid fibril composition determined. The type of ATTR was determined by Western blot in all but two patients. For these two patients a positive 99mTc-3,3-diphosphono-1,2-propanodicarboxylic acid scintigraphy indicated ATTR Type A.ResultsIn 14 of the 15 families, the same amyloid fibril composition was noted irrespective of differences in age at onset. In the one family, different ATTR fibril types was found in two brothers with similar ages at onset.ConclusionsFamily predisposition appears to have an impact on amyloid fibril composition in members of the family irrespective of their age at onset of disease, but if genetically determined, the gene/genes are likely to be situated at another location than the TTR gene in the genome.

Unusual duplication mutation in a surface loop of human transthyretin leads to an aggressive drug-resistant amyloid disease

Transthyretin (TTR) is a globular tetrameric transport protein in plasma. Nearly 140 single amino acid substitutions in TTR cause life-threatening amyloid disease. We report a one-of-a-kind pathological variant featuring a Glu51, Ser52 duplication mutation (Glu51_Ser52dup). The proband, heterozygous for the mutation, exhibited an unusually aggressive amyloidosis that was refractory to treatment with the small-molecule drug diflunisal. To understand the poor treatment response and expand therapeutic options, we explored the structure and stability of recombinant Glu51_Ser52dup. The duplication did not alter the protein secondary or tertiary structure but decreased the stability of the TTR monomer and tetramer. Diflunisal, which bound with near-micromolar affinity, partially restored tetramer stability. The duplication had no significant effect on the free energy and enthalpy of diflunisal binding, and hence on the drug-protein interactions. However, the duplication induced tryptic digestion of TTR at near-physiological conditions, releasing a C-terminal fragment 49-129 that formed amyloid fibrils under conditions in which the full-length protein did not. Such C-terminal fragments, along with the full-length TTR, comprise amyloid deposits in vivo. Bioinformatics and structural analyses suggested that increased disorder in the surface loop, which contains the Glu51_Ser52dup duplication, not only helped generate amyloid-forming fragments but also decreased structural protection in the amyloidogenic residue segment 25-34, promoting misfolding of the full-length protein. Our studies of a unique duplication mutation explain its diflunisal-resistant nature, identify misfolding pathways for amyloidogenic TTR variants, and provide therapeutic targets to inhibit amyloid fibril formation by variant TTR.

Positron emission tomography (PET) utilizing Pittsburgh compound B (PIB) for detection of amyloid heart deposits in hereditary transthyretin amyloidosis (ATTR)

BackgroundDPD scintigraphy has been advocated for imaging cardiac amyloid in ATTR amyloidosis. PET utilizing 11C-Pittsburgh compound B (PIB) is the gold standard for imaging brain amyloid in Alzheimer’s disease. PIB was recently shown to identify cardiac amyloidosis in both AL and ATTR amyloidosis. In the ATTR population, two types of amyloid fibrils exist, one containing fragmented and full-length TTR (type A) and the other only full-length TTR (type B). The aim of this study was to further evaluate PIB-PET in patients with hereditary ATTR amyloidosis. MethodsTen patients with biopsy-proven V30M ATTR amyloidosis and discrete or no signs of cardiac involvement were included. Patients were grouped according to TTR-fragmentation. All underwent DPD scintigraphy, echocardiography, and PIB-PET. A left ventricular PIB-retention index (PIB-RI) was established and compared to five normal volunteers. ResultsPIB-RI was increased in all patients (P < 0.001), but was significantly higher in type B than in type A (0.129 ± 0.041 vs 0.040 ± 0.006 min−1, P = 0.009). Cardiac DPD uptake was elevated in group A and absent in group B. ConclusionPIB-PET, in contrast to DPD scintigraphy, has the potential to specifically identify cardiac amyloid depositions irrespective of amyloid fibril composition. The heart appears to be a target organ for amyloid deposition in ATTR amyloidosis.

Positron Emission Tomography (PET) utilizing Pittsburgh compound B (PIB) detects amyloid heart deposits in hereditary transthyretin amyloidosis (ATTR).

Background DPD scintigraphy has been advocated for imaging cardiac amyloid in ATTR amyloidosis. PET utilizing 11C-Pittsburgh compound B (PIB) is the gold standard for imaging brain amyloid in Alzheimer’s disease. PIB was recently shown to identify cardiac amyloidosis in both AL and ATTR amyloidosis. In the ATTR population, two types of amyloid fibrils exist, one containing fragmented and full-length TTR (type A) and the other only full-length TTR (type B). The aim of this study was to further evaluate PIB-PET in patients with hereditary ATTR amyloidosis.

Liver transplantation in transthyretin amyloidosis: issues and challenges.

Hereditary transthyretin amyloidosis (ATTR) is a rare worldwide autosomal dominant disease caused by the systemic deposition of an amyloidogenic variant of transthyretin (TTR), which is usually derived from a single amino acid substitution in the TTR gene. More than 100 mutations have been described, with V30M being the most prevalent. Each variant has a different involvement, although peripheral neuropathy and cardiomyopathy are the most common. Orthotopic liver transplantation (OLT) was implemented as the inaugural disease-modifying therapy because the liver produces the circulating unstable TTR. In this review, we focus on the results and long-term outcomes of OLT for ATTR after more than 2063 procedures and 23 years of experience. After successful OLT, neuropathy and organ impairment are not usually reversed, and in some cases, the disease progresses. The overall 5-year survival rate is approximately 100% for V30M patients and 59% for non-ATTR V30M patients. Cardiac-related death and septicemia are the main causes of mortality. Lower survival is related to malnutrition, a longer duration of disease, cardiomyopathy, and a later onset (particularly for males). Deposits, which are composed of a mixture of truncated and full-length TTR (type A) fibrils, have been associated with posttransplant myocardial dysfunction. A higher incidence of early hepatic artery thrombosis of the graft has also been documented for these patients. Liver-kidney/heart transplantation is an alternative for patients with advanced renal disease or heart failure. The sequential procedure, in which ATTR livers are reused in patients with liver disease, reveals that neuropathy in the recipient may appear as soon as 6 years after OLT, and ATTR deposits may appear even earlier. Long-term results of trials with amyloid protein stabilizers or disrupters, silencing RNA, and antisense oligonucleotides will highlight the value and limitations of liver transplantation.

Specific Disruption of Transthyretin(105–115) Fibrilization Using “Stabilizing” Inhibitors of Transthyretin Amyloidogenesis

Transthyretin (TTR) is a cerebrospinal fluid and serum protein that undergoes ordered aggregation (amyloidogenesis) in familial amyloidotic polyneuropathy (FAP) and senile systemic amyloidosis (SSA). It is now widely accepted that dissociation of the native TTR tetramer is a precondition for amyloidogenesis; thus, molecules that stabilize the tetramer have received much attention as potential TTR amyloidosis inhibitors. Many of these inhibitors bind to the thyroxine (T(4)) binding pocket and interact specifically with a section of the TTR sequence, corresponding to residues 105-115, that is implicated in amyloidogenic propensity. In this work, we study the effects of "stabilizing" inhibitors on ordered aggregation of TTR(105-115) peptide. We show that molecules known to bind full-length TTR at the T(4) site are potent, specific inhibitors of ordered aggregation, while molecules that do not interact with TTR exhibit milder, nonspecific disruption through a "hyperbundling" effect. Our results suggest that, in addition to annealing the native tetramer, "stabilizing" inhibitors may also directly disrupt amyloidogenic aggregation of TTR monomers through specific interactions with the exposed TTR(105-115) sequence.

A Comprehensive Model for Packing and Hydration for Amyloid Fibrils of β2-Microglobulin

Volume can provide informative structural descriptions of macromolecules such as proteins in solution because a final volumetric outcome accompanies the exquisite equipoise of packing effects between residues, and residues and waters inside and outside proteins. Here we performed systematic investigations on the volumetric nature of the amyloidogenic conformations of beta2-microglobulin (beta2-m) and its amyloidogenic core peptide, K3, using a high precision densitometer. The transition from the acid-denatured beta2-m to the mature amyloid fibrils was accompanied by a positive change in the partial specific volume, which was larger than that observed for the transition from the acid-denatured beta2-m to the native structure. The data imply that the mature amyloid fibrils are more voluminous than the native structure because of a sparse packing density of side chains. In contrast, the formation of the mature amyloid-like fibrils of the K3 from the random coil was followed by a considerable decrease in the partial specific volume, suggesting a highly compact core structure. Interestingly, the immature amyloid-like fibrils of beta2-m exhibited a volume intermediate between those of the mature fibrils of beta2-m and K3, because of the core structure at their center and the relatively noncompact region around the core with much hydration. These volumetric differences would result from the nature of main-chain-dominated fibrillogenesis. We suggest comprehensive models for these three types of fibrils illustrating packing and hydrational states.

Transthyretin Interacts with Metallothionein 2

Transthyretin (TTR) is a 55 kDa homotetrameric protein known for the transport of thyroxine and the indirect transportation of retinol. Within the central nervous system, TTR is primary synthesized and secreted into the cerebral spinal fluid by the choroid plexus (CP), whereas most TTR in the systemic circulation is produced and secreted by the liver. TTR is involved in two types of amyloid disease, the senile systemic amyloidosis and the familial amyloidotic polyneuropathy. TTR has also been implicated in the sequestration of amyloid beta peptide (Abeta), preventing its deposition. To explore other biological roles for TTR, we searched for protein-protein interactions using the yeast two-hybrid system with the full-length human TTR cDNA as bait. We found a novel interaction between TTR and metallothionein 2 (MT2) in human liver. This interaction was confirmed by competition binding assays, co-immunoprecipitation, cross-linking, and Western blotting experiments. Binding studies using MT1 showed a saturable specific interaction with TTR with a Kd of 244.8 +/- 44.1 nM. Western blotting experiments revealed a TTR-MT1/2 protein complex present in rat CP and kidney tissue extracts. Immunofluorescence experiments, in CP primary cell cultures and in CP paraffin sections, showed co-localization of TTR and MT1/2 in the cytoplasm of epithelial CP cells and localization of MT1/2 in the endoplasmic reticulum. Moreover, dot blot immunoassays of rat CSF provided the first evidence, to our knowledge, of circulating metallothionein in CSF. Taken together, we suggest that TTR-MT1/2 complexes may be functionally significant not only in healthy conditions but also in Abeta deposition in Alzheimer disease, thereby providing a novel potential therapeutic target.

Detailed Structural Analysis of Amyloidogenic Wild-Type Transthyretin Using a Novel Purification Strategy and Mass Spectrometry

Wild-type transthyretin (TTR), normally a soluble plasma-circulating protein, can be amyloidogenic, i.e., form tissue-deposited fibrillar material in the extracellular matrix of various organs throughout the body. Senile systemic amyloidosis (SSA) is one such pathology and features TTR-containing amyloid deposits that are found primarily in the heart. The cause for this transition from soluble to insoluble protein in SSA is yet to be determined as specific structural features that might favor TTR fibrillogenesis have not yet been identified. The precise characterization of ex vivo fibril deposits might provide insight, but structural analyses of TTR from amyloid deposits have been hindered thus far by the lack of purification strategies that overcome the insolubility of the tissue-derived protein without degrading it. Consequently, the true biochemical nature of deposited TTR remains in question. In this study, we provide detailed analyses of both the soluble (serum) and deposited (tissue) forms of TTR from cases of SSA. In the serum, a distribution of mixed disulfides, specifically S-sulfonated and S-cysteinylated forms of TTR, as well as the unmodified protein were identified. The relative levels of the three TTR species in the SSA group were comparable to amounts present in sera from age-matched control groups. For characterization of the amyloid deposited TTR, we investigated cardiac tissue samples obtained from three separate cases of SSA. We report a novel chromatographic purification strategy performed under nonreducing conditions (to maintain cysteine disulfide status) and the use of this procedure in conjunction with detailed mass spectrometric analysis of TTR from the amyloid deposits. A series of C-terminal TTR fragments with N-termini ranging from amino acids 46 to 55 were identified. We also determined that the deposits in all samples contained Cys10 disulfide-linkedhomodimers composed of full-length TTR monomers. This last finding suggests an important role for Cys10 conjugation in the transition from soluble TTR to the pathological amyloid fibril.

Transthyretin-derived senile systemic amyloidosis: clinicopathologic and structural considerations

Senile systemic amyloidosis (SSA) is a prevalent disease affecting the elderly and results from the pathologic deposition, predominantly in the heart, of unmutated (wild-type) transthyretin (TTR) molecules. This disorder differs from the familial TTR-associated amyloidoses that generally occur in a younger population and involves peripheral nerves but, notably, the deposits contain mutated (variant) forms of the amyloidogenic precursor protein. To gain further insight into the clinicopathologic features and pathogenesis of SSA, we have reviewed the post-mortem findings of 33 Swedish individuals (27 men, 6 women) who had pronounced cardiac amyloid disease. Additionally, in all cases there were amyloid deposits within the lungs. Seven of the patients were uremic, possibly due to fibrillar deposits in the renal medullary papilla. In contrast to other systemic amyloidoses, none of the 33 had more than minute congophilic material in the renal cortex, liver, or spleen. We have also analyzed the chemical composition of fibrils extracted from myocardium and have found that the protein consisted mainly of C-terminal fragments of TTR starting at positions 46, 49, and 52. Furthermore, we identified through antigenic mapping, an epitope present on the H strand of fibrillar, but not normal, plasma TTR molecules indicating that Ibis strand is exposed in fibrils. We suggest that ATTR fibrils may develop both from full-length TTR and from C-terminal TTR fragments and that the N-terminal part is not necessary for the fibril integrity.