Selective silencing of a mutant transthyretin allele by small interfering RNAs

Familial amyloidotic polyneuropathy (FAP) is a hereditary systemic amyloidosis caused by dominantly acting missense mutations in the gene encoding transthyretin (TTR). The most common mutant TTR is of the Val30Met type, which results from a point mutation. Because the major constituent of amyloid fibrils is mutant TTR, agents that selectively suppress mutant TTR expression could be powerful therapeutic tools. This study has been performed to evaluate the use of small interfering RNAs (siRNAs) for the selective silencing of mutant Val30Met TTR in cell culture systems. We have identified an siRNA that specifically inhibits mutant, but not wild-type, TTR expression even in cells expressing both alleles. Thus, this siRNA-based approach may have potential for the gene therapy of FAP.     

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.     

Comparative studies of two transthyretin variants with protective effects on familial amyloidotic polyneuropathy: TTR R104H and TTR T119M

Recently, a new nonpathogenic transthyretin (TTR) variant-TTR R104H (TTR H104)-has been described in heterozygotic and compound heterozygotic individuals from a Japanese family with familial amyloidotic polyneuropathy (FAP). The compound heterozygotic individual, a carrier of TTR V30M (TTR M30) and TTR R104H (TTR M30/H104) presented a very mild form of FAP with slow progression of the disease. TTR and retinol binding protein (RBP) levels were found to be increased in serum from TTR H104 carriers. These characteristics are very similar to those found in compound heterozygotic carriers of TTR V30M-T119M (TTR M30/M119). To structurally compare these variants, we performed stability and thyroxine (T(4)) binding studies. TTR M30/H104 showed an increased resistance to dissociation into monomers similar to TTR M30/M119. This suggests that the His104 substitution has the same stabilizing effect on tetrameric TTR as the Met119 substitution. Concerning T(4) binding, TTR H104 presents a T(4) binding affinity lower than that of TTR M119, but still higher than normal TTR. However, TTR from the compound heterozygotic carrier of TTR M30/H104 presented a T(4) binding affinity lower than normal. The results indicate that the His 104 substitution induces structural alterations that increase the stability of the tetramer in compound heterozygotes for TTR M30 despite a lower affinity for T(4) binding. Thus, stability of TTR and binding affinity for T(4) may not be related. More detailed characterization of these variants is needed to clarify the structural alterations responsible for their increased stability.     

Cell based screening of inhibitors of transthyretin aggregation

The amyloidoses are the extracellular subset of a group of diseases in which in vivo protein misfolding leads to a pathologic gain of function, i.e., aggregation leading to protein deposition, with subsequent tissue damage. Wild-type and mutant transthyretins (TTR) are the etiologic agents in prototypic systemic amyloidoses. We describe a cell-based assay that measures the cytotoxicity of physiologic concentrations of the amyloidogenic Val30Met TTR variant (V30M TTR) using cells of the same lineage as the in vivo tissue target of amyloid deposition. We have utilized the assay to screen small molecules for their capacity to inhibit the TTR-induced cell damage. We compared the inhibitory activity of each compound with its ability to prevent TTR fibril formation in vitro. Our results emphasize the importance of screening compounds under physiologic conditions. Moreover, if a common conformational intermediate is responsible for cell death in all the amyloid diseases, the cell-based assay has the potential to aid in the discovery of compounds useful in the treatment of amyloidoses caused by other misfolded proteins as well as those caused by TTR.     

Comparative calorimetric study of non-amyloidogenic and amyloidogenic variants of the homotetrameric protein transthyretin

Familial amyloidotic polyneuropathy (FAP) is an autosomal dominant hereditary type of amyloidosis involving amino acid substitutions in transthyretin (TTR). V30M-TTR is the most frequent variant, and L55P-TTR is the variant associated with the most aggressive form of FAP. The thermal stability of the wild-type, V30M-TTR, L55P-TTR and a non-amyloidogenic variant, T119M-TTR, was studied by high-sensitivity differential scanning calorimetry (DSC). The thermal unfolding of TTR is a spontaneous reversible process involving a highly co-operative transition between folded tetramers and unfolded monomers. All variants of transthyretin are very stable to the thermal unfolding that occurs at very high temperatures, most probably because of their oligomeric structure. The data presented in this work indicated that for the homotetrameric form of the wild-type TTR and its variants, the order of stability is as follows: wild-type TTR approximately > T119M-TTR > L55P-TTR > V30M-TTR, which does not correlate with their known amyloidogenic potential.     

A specific test for transthyretin 122 (Val----Ile), based on PCR-primer-introduced restriction analysis (PCR-PIRA): confirmation of the gene frequency in blacks.

The variant transthyretin (TTR) allele, TTR (122 Val----Ile), associated with cardiac amyloidosis in blacks, is caused by a G----A transition which destroys a MaeIII site. This variant has previously been detected by PCR around codon 122, followed by MaeIII digestion, but this test is not specific: any of 12 mutations in the MaeIII recognition site, each of which yields a different amino acid change, would also destroy this site. A modification of PCR, termed "PCR-primer-introduced restriction analysis," was used to introduce a new FokI site into the PCR products derived from the variant (122 Ile) but not wild-type (122 Val) allele. This test demonstrated that each of six previously identified MaeIII(-) alleles had lost its MaeIII site because of a G----A transition encoding TTR (122 Val----Ile), confirming that the same TTR variant was present both in 4/177 healthy black individuals and as a homozygous variant in an individual with cardiac amyloidosis.     

Haplotype analysis of common transthyretin mutations

The most frequent transthyretin (TTR) variant associated with hereditary amyloidosis is TTR Met 30, which has its major focus in Portugal, although it also occurs in many other countries. The distribution of the mutation and its occurrence in a CpG dinucleotide lead us to question the origin of the mutation and the possibility of its having originated in Portugal. In order to investigate these questions, we studied the distribution of haplotypes associated with the Met 30 mutation in families from different European countries. All the analysed Portuguese families presented the same haplotype associated with the Met 30 mutation (haplotype I). The same was found for the Swedish and Spanish families studied. However, a distinct haplotype (haplotype III) was found in three families, one Italian, one English and one Turkish. These results suggest that, although the Portuguese Met 30 carriers might have one founder, the mutation probably recurred in populations in Europe in a similar manner to that reported in Japan. In this study, we have also analysed the haplotypes associated with other TTR variants frequent in the Portuguese population.     

Production and functional analysis of normal and variant recombinant human transthyretin proteins.

The most common form of hereditary systemic amyloidosis is familial amyloidotic polyneuropathy associated with single amino acid changes in the plasma protein, transthyretin. In addition, there are two variants of transthyretin (Ser6 and Thr109) not associated with familial amyloidotic polyneuropathy but with familial euthyroid hyperthyroxinemia, also an autosomal dominant disorder. In these autosomal dominant diseases, most affected individuals are heterozygous and therefore have hybrid forms of the tetrameric plasma transthyretin. In order to study the structure/function relationships of homozygous variant transthyretins, normal human transthyretin and five variant transthyretins (Gly6----Ser, Leu58----His, Thr60----Ala, Ile84----Ser, and Ala109----Thr) were produced in Escherichia coli using the expression vector, pCZ11, and site-directed mutagenesis. These recombinant transthyretin (r-TTR) proteins showed the correct size (14 kilodaltons) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western analysis and self-associated into tetramers as determined by size exclusion chromatography. Recombinant normal, Ser6, and Ala60 r-TTRs had an affinity for thyroxine indistinguishable from normal human TTR purified from plasma, whereas His58 and Ser84 r-TTRs had significantly reduced affinity. On the other hand, Thr109 r-TTR had a much higher affinity, probably due to its position within the thyroxine-binding pocket. Expression of mutant transthyretins in E. coli provides the opportunity to study structure/function relationships and amyloid-forming capabilities induced by single amino acid substitutions in the transthyretin molecule.     

Why is Leu55-->Pro55 transthyretin variant the most amyloidogenic: insights from molecular dynamics simulations of transthyretin monomers

Transthyretin (TTR) is one of the known human amyloidogenic proteins. Its native state is a homotetramer with each monomer having a beta-sandwich structure. Strong experimental evidence suggests that TTR dissociates into monomeric intermediates and that the monomers subsequently self-assemble to form amyloid deposits and insoluble fibrils. However, details on the early steps along the pathway of TTR amyloid formation are unclear, although various experimental approaches with resolutions at the molecular or residue level have provided some clues. It is highly likely that the stability and flexibility of monomeric TTR play crucial roles in the early steps of amyloid formation; thereby, it is essential to characterize initial conformational changes of TTR monomers. In this article we probe the possibility that the differences in the monomeric forms of wild-type (WT) TTR and its variants are responsible for differential amyloidogenesis. We begin with the simulations of WT, Val30-->Met (V30M), and Leu55-->Pro (L55P) TTR monomers. Nanosecond time scale molecular dynamics simulations at 300 K were performed using AMBER. The results indicate that the L55P-TTR monomer undergoes substantial structural changes relative to fluctuations observed in the WT and V30M TTR monomers. The observation supports earlier speculation that the L55P mutation may lead to disruption of the beta-sheet structure through the disorder of the "edge strands" that might facilitate amyloidogenesis.     

Specific pathogen free conditions prevent transthyretin amyloidosis in mouse models

Transthyretin (TTR) associated amyloidosis is an autosomal dominant disorder characterized by peripheral and autonomic neuropathy. Both genetic and environmental factors are thought to be involved in development of TTR associated amyloidosis. Previously, we demonstrated that amyloid deposition was observed in various tissues of transgenic mouse lines carrying a human mutant TTR (Met30) gene. To analyze the influence of environmental factors on TTR amyloidosis, these amyloidogenic transgenic mouse models were kept under conventional (CV) or specific pathogen free (SPF) conditions. Although the serum levels of Met30 for mice housed in the CV and SPF conditions were similar, amyloid deposition was observed in CV conditions, but not in SPF conditions. In addition, the extent of amyloid deposition in transgenic mice was dependent on duration kept under CV conditions. There were significant differences in proportion of amyloid deposition in several tissues between CV and SPF conditions. Maintenance of these mice at 30 degrees C did not induce amyloid deposition in SPF conditions. These results suggest that the SPF conditions can completely prevent amyloid deposition, and that environmental factors can affect the onset and progression even in a single gene disorder.     

Study of an anti-human transthyretin immunoadsorbent: Influence of coupling chemistry on binding capacity and ligand leakage

A variant of transthyretin (TTR Val30Met) has been identified as the main protein precursor of the amyloid fibrils deposited in familial amyloidotic polyneuropathy (FAP). Specific removal of TTR in an extracorporeal immunoadsorption procedure is currently under investigation as a possible treatment of FAP. Immunoadsorbents were constructed by immobilizing murine anti-TTR monoclonal antibody 88.6.BA9 onto agarose gel supports via several different coupling chemistries. The influence of coupling conditions such as pH and antibody density, and of perfusion variables, such as antigen concentration and applied flow-rate, on the TTR capture efficiency, was determined. Cyanogen bromide-, carbonyldiimidazole- and aldehyde-activated (ALD) supports conjugated with antibody at optimal pH, provided immunoadsorbents with comparable TTR binding capacities. Regarding stability, leakage was lowest for the ALD based immunoadsorbents, particularly at high pH.     

Hepatic production of transthyretin L12P leads to intracellular lysosomal aggregates in a new somatic transgenic mouse model

Transthyretin (TTR) is a plasma and cerebrospinal fluid (CSF)-circulating homotetrameric protein. More than 100 point mutations have been identified in the TTR gene and several are related with amyloid diseases. Here we focused our attention in the TTR L12P variant associated with severe peripheral neuropathy and leptomeningeal amyloidosis. By using different cell lines derived from tissues specialized on TTR synthesis, such as the hepatocyte and the choroid plexus expressing WT, V30M, or L12P TTR variants we analyzed secretion, intracellular aggregation and degradation patterns. Also, we used liver-specific AAV gene transfer to assess expression of the L12P variant in vivo. We found the following: (i) decreased secretion with intracellular aggregation of TTR L12P in hepatoma cells relative to WT and V30M variant; this differential property of TTR L12P variant was also observed in mice injected with L12P AAV vector; (ii) differential N-glycosylation pattern of L12P variant in hepatoma cell lysates, conditioned media and mouse sera, which might represent an escape mechanism from ERAD degradation; (iii) intracellular L12P TTR aggregates mainly localized to lysosomes in cultured cells and liver; and (iv) none of the above findings were present in choroid plexus derived cells, suggesting particular secretion/quality control mechanisms that might contribute to leptomeningeal amyloidosis associated with the L12P variant. These observations open new avenues for the treatment of TTR associated leptomeningeal amyloidosis.     

D18G transthyretin is monomeric,aggregation prone,and not detectable in plasma and cerebrospinal fluid: a prescription for central nervous system amyloidosis?

Over 70 transthyretin (TTR) mutations facilitate amyloidosis in tissues other than the central nervous system (CNS). In contrast, the D18G TTR mutation in individuals of Hungarian descent leads to CNS amyloidosis. D18G forms inclusion bodies in Escherichia coli, unlike the other disease-associated TTR variants overexpressed to date. Denaturation and reconstitution of D18G from inclusion bodies afford a folded monomer that is destabilized by 3.1 kcal/mol relative to an engineered monomeric version of WT TTR. Since TTR tetramer dissociation is typically rate limiting for amyloid formation, the monomeric nature of D18G renders its amyloid formation rate 1000-fold faster than WT. It is perplexing that D18G does not lead to severe early onset systemic amyloidosis, given that it is the most destabilized TTR variant characterized to date, more so than variants exhibiting onset in the second decade. Instead, CNS impairment is observed in the fifth decade as the sole pathological manifestation; however, benign systemic deposition is also observed. Analysis of heterozygote D18G patient's serum and cerebrospinal fluid (CSF) detects only WT TTR, indicating that D18G is either rapidly degraded postsecretion or degraded within the cell prior to secretion, consistent with its inability to form hybrid tetramers with WT TTR. The nondetectable levels of D18G TTR in human plasma explain the absence of an early onset systemic disease. CNS disease may result owing to the sensitivity of the CNS to lower levels of D18G aggregate. Alternatively, or in addition, we speculate that a fraction of D18G made by the choroid plexus can be transiently tetramerized by the locally high thyroxine (T(4)) concentration, chaperoning it out into the CSF where it undergoes dissociation and amyloidogenesis due to the low T(4) CSF concentration. Selected small molecule tetramer stabilizers can transform D18G from a monomeric aggregation-prone state to a nonamyloidogenic tetramer, which may prove to be a useful therapeutic strategy against TTR-associated CNS amyloidosis.     

Monoaryl derivatives as transthyretin fibril formation inhibitors: Design,synthesis, biological evaluation and structural analysis

Transthyretin (TTR) is a ß-sheet-rich homotetrameric protein that transports thyroxine (T4) and retinol both in plasma and in cerebrospinal fluid. TTR also interacts with amyloid-β, playing a protective role in Alzheimer’s disease. Dissociation of the native transthyretin (TTR) tetramer is widely accepted as the critical step in TTR amyloids fibrillogenesis, and is responsible for extracellular deposition of amyloid fibrils. Small molecules, able to bind in T4 binding sites and stabilize the TTR tetramer, are interesting tools to treat and prevent systemic ATTR amyloidosis. We report here the synthesis, in vitro evaluation and three-dimensional crystallographic analyses of new monoaryl-derivatives in complex with TTR. Of the derivatives reported here, the best inhibitor of TTR fibrillogenesis, 1d, exhibits an activity similar to diflunisal.     

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.     

The A-chain of insulin contacts the insert domain of the insulin receptor. Photo-cross-linking and mutagenesis of a diabetes-related crevice

The contribution of the insulin A-chain to receptor binding is investigated by photo-cross-linking and nonstandard mutagenesis. Studies focus on the role of Val(A3), which projects within a crevice between the A- and B-chains. Engineered receptor alpha-subunits containing specific protease sites ("midi-receptors") are employed to map the site of photo-cross-linking by an analog containing a photoactivable A3 side chain (para-azido-Phe (Pap)). The probe cross-links to a C-terminal peptide (residues 703-719 of the receptor A isoform, KTFEDYLHNVVFVPRPS) containing side chains critical for hormone binding (underlined); the corresponding segment of the holoreceptor was shown previously to cross-link to a Pap(B25)-insulin analog. Because Pap is larger than Val and so may protrude beyond the A3-associated crevice, we investigated analogs containing A3 substitutions comparable in size to Val as follows: Thr, allo-Thr, and alpha-aminobutyric acid (Aba). Substitutions were introduced within an engineered monomer. Whereas previous studies of smaller substitutions (Gly(A3) and Ser(A3)) encountered nonlocal conformational perturbations, NMR structures of the present analogs are similar to wild-type insulin; the variant side chains are accommodated within a native-like crevice with minimal distortion. Receptor binding activities of Aba(A3) and allo-Thr(A3) analogs are reduced at least 10-fold; the activity of Thr(A3)-DKP-insulin is reduced 5-fold. The hormone-receptor interface is presumably destabilized either by a packing defect (Aba(A3)) or by altered polarity (allo-Thr(A3) and Thr(A3)). Our results provide evidence that Val(A3), a site of mutation causing diabetes mellitus, contacts the insert domain-derived tail of the alpha-subunit in a hormone-receptor complex.     

Functional consequences of mutational analysis of norovirus protease

Norovirus protease has been subjected to an extensive mutagenesis study. Ala-scanning mutation at 13 different positions (Trp6, Trp19, Thr27, Leu86, Leu95, Leu97, Met101, Gln117, Leu121, Thr134, Tyr143, Val144, and Val167) led to loss of function and/or stability. Considering the crystal structure of the protease, it was revealed that a hydroxyl group of Thr134 and an aromatic ring of Tyr143 were important for substrate recognition along with His157. It was notable that several of the residues identified were in close proximity to each other, suggesting their importance for the integrity and stability of the protease.     

Identification of two different point mutations associated with the fluoride-resistant phenotype for human butyrylcholinesterase.

The fluoride variant of human butyrylcholinesterase owes its name to the observation that it is resistant to inhibition by 0.050 mM sodium fluoride in the in vitro assay. Individuals who are heterozygous for the fluoride and atypical alleles experience about 30 min of apnea, rather than the usual 3-5 min, after receiving succinyldicholine. Earlier we reported that the atypical variant has a nucleotide substitution which changes Asp 70 to Gly. In the present work we have identified two different point mutations associated with the fluoride-resistant phenotype. Fluoride-1 has a nucleotide substitution which changes Thr 243 to Met (ACG to ATG). Fluoride-2 has a substitution which changes Gly 390 to Val (GGT to GTT). These results were obtained by DNA sequence analysis of the butyrylcholinesterase gene after amplification by PCR. The subjects for these analyses were 4 patients and 21 family members.     

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.     

Oxidation inhibits amyloid fibril formation of transthyretin

The role of amino acid side chain oxidation in the formation of amyloid assemblies has been investigated. Chemical oxidation of amino acid side chains has been used as a facile method of introducing mutations on protein structures. Oxidation promotes changes within tertiary contacts that enable identification of residues and interactions critical in stabilizing protein structures. Transthyretin (TTR) is a soluble human plasma protein. The wild-type (WT) and several of its variants are prone to fibril formation, which leads to amyloidosis associated with many clinical syndromes. The effects of amino acid side chain oxidations were investigated by comparing the kinetics of fibril formation of oxidized and unoxidized proteins. The WT and V30M TTR mutant (valine 30 substituted with methionine) were allowed to react over a time range of 10 min to 12 h with hydroxy radical and other reactive oxygen species. In these timescales, up to five oxygen atoms were incorporated into WT and V30M TTR proteins. Oxidized proteins retained their tetrameric structures, as determined by cross-linking experiments. Side chain modification of methionine residues at position 13 and 30 (the latter for V30M TTR only) were dominant oxidative products. Mono-oxidized and dioxidized methionine residues were identified by radical probe mass spectometry employing a footprinting type approach. Oxidation inhibited the initial rates and extent of fibril formation for both the WT and V30M TTR proteins. In the case of WT TTR, oxidation inhibited fibril growth by approximately 76%, and for the V30M TTR by nearly 90%. These inhibiting effects of oxidation on fibril growth suggest that domains neighboring the methionine residues are critical in stabilizing the tetrameric and folded monomer structures.