Calcium Binding Proteins (Wiley Series in Protein and Peptide Science)

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Therefore, we assayed the ability of T 4 to bind to TTR in the plasma of the heterozygote subject carrying the AV mutation. Plasma was incubated with [ I]-labelled T 4 , followed by separation of T 4 binding proteins using native gel electrophoresis and T 4 binding proteins visualization by autoradiography. The intensity of the bands corresponding to TTR in the different plasma samples was determined by densitometry. Control plasma control was used as reference for migration. All samples were run in the same gel though not sequentially. The place where the gel was trimmed is indicated.

In order to attain a more precise characterization of T 4 binding properties, a competition assay using gel filtration chromatography, was performed. In this assay, a constant amount of each TTR variant was incubated with a small amount of [ I]T 4 and with increasing concentrations of non-labelled T 4 as competitor. T 4 bound to TTR was separated from the unbound molecule in the size exclusion column. The results were expressed as percentage of binding against logarithm of the concentration of non-labelled T 4. The obtained competition curves are presented in Fig.

In good agreement with the electrophoretic data, the assay clearly demonstrates that the mutant TTR variants do not exhibit T 4 binding affinity as no competition was observed, in contrast to what happens for TTR WT and TTR TM, that were tested in parallel.

Pathogenic mutations associated with inherited TTR amyloidosis make the protein more susceptible to aggregation. To date, it is still not completely clear how TTR amyloid formation occurs under physiological conditions, but the most accepted hypothesis assumes that mutations in TTR impact the structural equilibrium of the protein, destabilizing the tetramer in favour of dissociated intermediates, which exhibit amyloidogenic potential.

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The age of onset and the penetrance of the disease depend on the specific mutation. As a general trend, the more destabilizing the mutation is, the more pronounced the amyloidogenicity of the protein variant. It is worth to point out here that an alternative mechano-enzymatic cleavage mechanism has been recently proposed to underlie transthyretin amyloidogenesis in vivo According to this new view, the combined effect of proteolysis and shearing would result in the release of a highly amyloidogenic 49— truncated protomer, usually found in ex vivo deposits.

The number of non-amyloidogenic mutations identified so far for TTR is small 44 , which is consistent with the idea that most sporadic mutations in proteins have a destabilizing effect Some mutations, such as G6S 46 or H90N 47 have a negligible impact on TTR stability and are considered non-pathogenic, whereas others like RH 48 or TM 30 have been shown to act as trans-suppressors. Trans-suppressor mutations render TTR more stable against dissociation and aggregation, reducing the symptoms of the disease in heterozygotes carrying additional aggressive mutations in the other allele. The trans-suppressor effects of RH and TM respond to different mechanisms The quaternary structure of RH is thermodynamically stabilised, whereas the tetrameric native state of TM exhibits a high kinetic stability.

The stabilizing effect of TM mutation is much higher than that of RH, which only modestly protects against aggregation. Thr protrudes directly into the T 4 binding site and the high kinetic stabilization provided by its mutation into Met owes to the novel contacts that this hydrophobic residue establishes or induces between amino acids belonging to different dimers, which strengthen the interaction between dimers at the T 4 binding pocket.

This is the same principle exploited by hundreds of small compounds that by binding to and filling the two T 4 binding sites stabilize the native state of TTR over the dissociative transition state. Thus, reinforcing the contacts at the quaternary dimer-dimer interface appears as a requirement to impose kinetic stability on the entire TTR tetramer.

It is worth to emphasize here that TM TTR is also totally resistant to proteolytic cleavage under shearing Here we report on a novel mutation affecting residue Ala that as Thr is located in the T 4 -binding cavity. Mutations of the adjacent Ala residue to Thr 49 or Val 50 are non-amyloidogenic, whereas mutation to Ser is pathogenic 51 ; however, Ala does not face the T 4 binding cavity.

The subject bearing the mutation AV in heterozygoty presented a mild, non-progressive, sensory neuropathy without any dysautonomic symptom or sign, a course that is not typical of a TTR-related polyneuropathy. Since it was not possible to follow-up the patient for more than 3 years, and there was almost no progression of disease, and no family history was available, the atypical picture suggests an idiopathic neuropathy or, more interesting, that the novel mutation is associated with a very benign expression of TTR-associated disease.

Globally, the AV mutation adds four additional methylene groups, two at each of the T 4 -binding sites at the dimer-dimer interface. This is important, because experiments with small molecules demonstrate that their binding to the first and second T 4 -binding sites additively increase the activation barrier associated with tetramer dissociation.

The generated structural model suggested that the interface would still admit four additional methylenes in the form of an AI mutation, without introducing steric clashes that might result in strain at the interface. Their high stabilities in front of urea denaturation at equilibrium and, specially, the very low dissociation rates they exhibit under highly denaturing conditions, are consistent with the existence of a high kinetic barrier for the dissociation TTR AV and TTR AI homotetramers. HHP causes denaturation of proteins by promoting the entrance of water molecules into the protein cavities, facilitating the population of partially folded states 52 , which in TTR, affects the dissociation of the tetramer.

The occupation of the T 4 -binding sites by the additional methylene groups of Val and Ile makes these variants, specially TTR AI, resistant to pressure. This effect may respond either to a stabilization of the ground state of the protein or to a destabilization of the dissociative transition state.

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In any case, it results in a dramatic kinetic stabilization for these proteins, which is translated in almost a complete suppression of their aggregation under acidic conditions. Interestingly enough, no mutation has previously been found in position of TTR. Thus, our data provide yet another piece of the puzzle in elucidating the correlation between mutations and aggregation propensity in the context of TTR amyloidoses.

The almost solved X-ray structures of TTR WT and its variants have not revealed major conformational changes that could explain their differential amyloidogenicity 53 , They are rather small differences at specific positions that account for changes in protein stability 19 , 41 , Thus, the quaternary structure of the mutant variants is assembled by additional new inter-subunit contacts that might account for their resistance to dissociation.

This destabilization has been attributed to the fact that Leu is in the dimer-dimer interface and the substitution will reduce the contact area. These contacts, absent or weak in TTR WT, repack the hydrophobic core of the T 4 -binding cavity stabilizing the tetramer. This explains why an increase in the hydrophobic volume of the residue in position results in a higher stabilizing effect than when the same type of change occurs in position Indeed, all the mutations described in this loop are linked to disease.

α-Helical Protein Assembly Motifs

It is worth to emphasize, that trans-suppressor mutations of TTR are of considerable interest, because they can be potentially employed to treat TTR-related amyloid diseases using liver targeted gene therapy. TTR AI, is perhaps the most stable mutant attained so far with a single amino acid change not involving covalent cross-linking.

Indeed, small molecules designed to impair TTR aggregation exert their stabilizing effect by binding with high affinity to the T 4 -binding site, thus competing the binding of the hormone. The novel contacts established by the Val and Ile side chains inside the T 4 -binding cavity should turn to be useful to engineer barrier heights in kinetic stabilizers by functionalizing them with chemical groups that by mimicking those interactions would strengthen the contacts at the dimer-dimer interface to the level observed in these two highly stable, non-amyloidogenic mutants.

A year-old healthy woman reported a months history of insidious onset and mild progression of symmetrical numbness of the feet with painful dysaesthesia. Pain sensation was slightly reduced below ankles. Vibration and position sensation were normal. Ankle reflexes were weak, otherwise tendon reflexes were normal.

Ataxia was absent and muscle strength was normal. There was no evidence of autonomic nerve or pyramidal tract dysfunction. There was no previous history of diabetes, alcohol intake or drug toxicity.

Family history was negative for polyneuropathy or other neurological disorders. Nerve conduction studies confirmed mild sensory neuropathy, with absent sural nerve action potential on both sides, but normal sensory potentials in upper limbs radial and median, bilaterally and unremarkable motor nerve conduction velocities and F-waves median and peroneus, bilaterally.

The following blood tests were normal or negative: full blood count, glycaemia, liver and renal function tests, thyroid hormone and vitamin B12 levels, serum immunoglobulins, autoantibody testing for anti-nuclear, extractable nuclear antigen and anti-neutrophil cytoplasmic antibodies, hepatitis C and HIV-1 serology.

Over the following three years the patient was observed every 6-months and a comparative nerve conduction study was repeated every year, without any evidence of disease progression. Following this period the patient was lost for follow-up.

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Blood samples were collected according to standards established by the latest revision of the Declaration of Helsinki. All methods were performed in accordance with the relevant guidelines and regulations. Informed consent was obtained from all donors in this study, including the propositus.

Calcium Binding Proteins (Wiley Series in Protein and Peptide Science) Calcium Binding Proteins (Wiley Series in Protein and Peptide Science)
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