Tetramer Dissociation and Monomer Partial Unfolding Precedes Protofibril Formation in Amyloidogenic Transthyretin Variants

Abstract

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.

Potentially amyloidogenic conformational intermediates populate the unfolding landscape of transthyretin: Insights from molecular dynamics simulations

“…The conformational stability of monomeric forms of WT-, L55P-, V30M-, and T119M-TTR was previously measured in our group through chemically-induced unfolding experiments followed by intrinsinc tryptophan fluorescence. 8 The analysis of structural properties extracted from the simulations, reported in the previous sections, show consistency with this previous experimental work. In fact, the variation of all analysed properties, but in particular C a -RMSD (Fig.…”

“…These results are in agreement with the hypothesis that residue Trp79 is the main responsible for the fluorescence increase observed in the chemically-induced unfolding experiments. 8 Thus, the reduced propensity of L55P-TTR to retain the initial native structure, as seen in the simulations, correlates well with the experimental evidence showing its lower monomer conformational stability 8 and higher tendency to form a larger amount of partially unfolded monomeric species. 11 Comparison with H/D exchange studies by NMR Wemmer and coworkers, using H/D exchange studies followed by NMR, determined solvent protection factors for WT-and L55P-TTR.…”

“…Employing classical and geometry-based methods as well as several property-fluctuation analyses and cluster analysis, we shed new light on the molecular mechanism of amyloidogenesis by TTR and extend the observations by other authors. [31][32][33][34] Our simulations corroborate experimental observations correlating the conformational stability of monomeric TTR species and their amyloidogenic potential, 8 and highlight some of the amino acid residues that may have a relevant role in the early stages of amyloid formation. Additionally, our simulations allow the identification of unfolding events leading to aggregationprone intermediates, and thus allow the discrimination between amyloidogenic and nonamyloidogenic behavior.…”

“…7) clearly indicate a higher tendency for unfolding of the L55P-TTR monomer when compared with the wild type, agreeing with the lower experimentally determined conformational stability of the L55P-TTR monomer. 8 Interestingly, while the side-chain SASA of residue Trp41 changes erratically across all simulations, Trp79, belonging to the helical motif and hidden from the solvent in the native state, appears more exposed in all simulations of L55P-TTR, with an overall average SASA of 26.9 6 24.8 Å 2 , than in those of WT-TTR, with an average SASA of 11.8 6 17.6 Å 2 . These results are in agreement with the hypothesis that residue Trp79 is the main responsible for the fluorescence increase observed in the chemically-induced unfolding experiments.…”

“…Experimental evidences based on aging experiments of tetrameric TTR and chemically induced unfolding experiments, revealed that tetramer dissociation into compact monomers with low conformational stability precedes amyloid fibril formation. 8 It has also become apparent that amyloidogenic single-point mutations influence not only TTR dissociation kinetics 9 but yield monomers with lower thermodynamic stability, 8 in spite of the high structural similarity between the mutants and the wildtype protein. 7 Additionally, amyloidogenic TTR variants were shown to produce, in solution and even at neutral pH, higher amounts of partially unfolded monomeric species with enhanced propensity for ordered aggregation into amyloid fibrils.…”