Characterization of the Transthyretin Acid Denaturation Pathways by Analytical Ultracentrifugation:  Implications for Wild-Type, V30M, and L55P Amyloid Fibril Formation

Abstract

Analytical ultracentrifugation methods were utilized to further characterize the acid denaturation pathways of wild-type, V30M, and L55P transthyretin (TTR) that generate intermediates leading to amyloid fibril formation and possibly the diseases senile systemic amyloidosis and familial amyloid polyneuropathy. Equilibrium and velocity methods were employed herein to characterize the TTR quaternary structural requirements for amyloid fibril formation. From neutral to slightly acidic conditions (pH 7.5-5.1), wildtype transthyretin (0.2-0.3 mg/mL, 100 mM KCl, 37°C) exists as a tetramer and is incapable of fibril formation. Under more acidic conditions (pH 5 to 3.9), tetrameric wild-type TTR slowly dissociates to a monomer having an alternatively folded tertiary structure(s) that self-assembles at physiological concentration (0.2 mg/mL) into a ladder of quaternary structural intermediates of increasing molecular weight. These intermediates appear to be on the pathway of amyloid fibril formation, since they ultimately disappear when amyloid fibrils are observed. The V30M and L55P TTR variants exhibit similar acid denaturation pathways, with the exception that dissociation of the tetramer to the monomeric amyloidogenic intermediate occurs at a higher pH and to a much greater extent, allowing the quaternary structural intermediates to be readily observed by velocity methods. Partial denaturation and assembly of the monomeric amyloidogenic intermediate(s) occur at pH 5.4 for V30M and L55P TTR over a 72 h period, during which wild-type TTR maintains its normal tetrameric three-dimensional structure. Interestingly, the L55P and V30M familial amyloid polyneuropathy (FAP) associated variants form amyloid protofilaments at pH 7.5 (37°C) after several weeks of incubation, suggesting that the activation barriers for TTR tetramer dissociation to the monomeric amyloidogenic intermediate are much lower for the FAP variants relative to wild-type TTR, which does not form amyloid or amyloid protofilaments under these conditions. This study establishes the key role of the monomeric amyloidogenic intermediate and its selfassembly into a ladder of quaternary structural intermediates for the formation of wild-type, V30M, and L55P transthyretin amyloid fibrils.

Synthesis, Structure, and Activity of Diclofenac Analogues as Transthyretin Amyloid Fibril Formation Inhibitors

“…Consistent with this, L55P TTR is markedly less stable to acid-induced tetramer dissociation and amyloid formation than V30M TTR, which is in turn less stable than wild type TTR. 11,[19][20][21] Thus, for these three variants, the ages of onset of TTR amyloid diseases correlate directly with the acid stabilities of the TTR tetramers involved.…”

“…7 Under acidic conditions, like those found in cellular organelles such as the lysosome, TTR dissociates to an alternatively folded, monomeric intermediate that self-assembles into amyloid fibrils. [8][9][10][11][12] The deposition of wild type TTR amyloid has been implicated to cause the disease senile systemic amyloidosis (SSA), whereas the deposition mutant TTR amyloid has been implicated to cause the diseases familial amyloid cardiomyopathy (FAC) and familial amyloid polyneuropathy (FAP); over 80 variants of TTR have been associated with FAC and FAP. 13,14 SSA involves the deposition of amyloid in the heart, with symptoms typically appearing around ages 70 to 80.…”

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“…Understanding the conditions that promote the accumulation of amyloidogenic intermediates, as well as their underlying common structural features, should provide further information about amyloidogenesis in general. Recent biophysical studies on TTR have identified in vitro conditions that stimulate amyloid formation 9,10,20 ( Fig. 1).…”

“…However, over the pH range of 5.0-3.9, TTR dissociates to a monomer that exhibits an altered but defined tertiary structure, as probed by fluorescence and far and near UV circular dichroism. The extent of amyloidogenesis correlates with the concentration of amyloidogenic monomer, which is maximal at pH 4.4 9,10 . Upon further acidification (< pH 3.9), the structurally defined monomers adopt alternative conformations analogous to a molten globule-like aciddenatured state (A-state), which forms low molecular weight A-state aggregates but not amyloid fibrils 21 .…”

“…Previous thinking about structural changes in the amyloidogenic intermediate, based on low resolution spectroscopic data, suggested that TTR amyloidogenesis begins with dissociation of tetramer followed by separation of β-strands C and D from the core of the monomer 9,10,14,19,27 . Our study reveals that all hydrogen bonds connecting β-strands B and C are destabilized, while those connecting β-strands D and A remain partially intact.…”