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.
Palhano
Fernando L
FL
Instituto de Bioquímica Médica, Programa de Biologia Estrutural, and Instituto Milênio de Biologia Estrutural e Biotecnologia Universidade Federal do Rio de Janeiro, Rio de Janeiro, 21941-590, Brazil.
Leme
Larissa P
LP
Busnardo
Roberta G
RG
Foguel
Debora
D
D019943:Amino Acid Substitution; D028227:Amyloid Neuropathies, Familial / Q000134:cerebrospinal fluid / Q000235:genetics / Q000628:therapy*; D006801:Humans; D016031:Liver Transplantation; D020125:Mutation, Missense; D011228:Prealbumin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000627:therapeutic use; D017510:Protein Folding; D020836:Protein Structure, Quaternary / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics
D011228:Prealbumin