Tetramer dissociation and monomer partial unfolding precedes protofibril formation in amyloidogenic transthyretin variants

Amyloid fibril formation and deposition is a common feature of a wide range of fatal diseases including spongiform encephalopathies, Alzheimer's disea se, and familial amyloidotic polyneuropathies (FAP), among many others. In certain forms of FAP, the amyloid fibrils are mostly constituted by variant s of transthyretin (TTR), a homotetrameric plasma protein. Recently, we sho wed 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-nat ive monomer is a compact structure, does not behave as a molten globule, an d may lead to the formation of partially unfolded monomeric species and hig h molecular mass soluble aggregates (Quintas, A., Saraiva, M.J.M., and Brit o, R.M.M. (1999) J. Biol. Chem. 274, 32943-32949), Here, based on aging exp eriments of tetrameric TTR and chemically induced protein unfolding experim ents 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-nat ive monomer produce the largest amount of partially unfolded monomeric spec ies and soluble aggregates under conditions that are close to physiological . Additionally, the soluble aggregates formed by the amyloidogenic TTR vari ants showed morphological and thioflavin-T fluorescence properties characte ristic 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 o riginates partially unfolded monomeric species with a high tendency for ord ered aggregation into amyloid fibrils. Thus, partial unfolding and conforma tional fluctuations of molecular species with marginal thermodynamic stabil ity may play a crucial role on amyloid formation in vivo.