Designing transthyretin mutants affecting tetrameric structure: implications in amyloidogenicity.
The molecular mechanisms that convert soluble transthyretin (TTR) tetramers into insoluble amyloid fibrils are still unknown; dissociation of the TTR tetramer is a pre-requisite for amyloid formation in vitro and involvement of monomers and/or dimers in fibril formation has been suggested by structural studies. We have designed four mutated proteins with the purpose of stabilizing [Ser(117)-->Cys (S117C) and Glu(92)-->Cys (E92C)] or destabilizing [Asp(18)-->Asn (D18N) and Leu(110)-->Ala (D110A)] the dimer/tetramer interactions in TTR, aiming at elucidating structural determinants in amyloidogenesis. The resistance of the mutated proteins to dissociation was analysed by HPLC studies of diluted TTR preparations. Both 'stabilized' mutants migrated as tetramers and, upon dilution, no other TTR species was observed, confirming the increased resistance to dissociation. For the 'destabilized' mutants, a mixture of tetrameric and monomeric forms co-existed at low dilution and the latter increased upon 10-fold dilution. Both of the destabilizing mutants formed amyloid in vitro when acidified. This result indicated that both the AB loop of TTR, destabilized in D18N, and the hydrophobic interactions affecting the dimer-dimer interfaces in L110A are implicated in the stability of the tetrameric structure. The stabilized mutants, which were dimeric in nature through disulphide bonding, were unable to polymerize into amyloid, even at pH 3.2. When the amyloid formation assay was repeated in the presence of 2-mercaptoethanol, upon disruption of the S-S bridges of these stable dimers, amyloid fibril formation was observed. This experimental evidence suggests that monomers, rather than dimers, are the repeating structural subunit comprising the amyloid fibrils.
Redondo
C
C
Amyloid Unit, Institute for Molecular and Cell Biology, University of Porto, Portugal.
Damas
A M
AM
Saraiva
M J
MJ
D017772:Amyloid Neuropathies / Q000235:genetics; D001704:Biopolymers / Q000737:chemistry; D019281:Dimerization; D004926:Escherichia coli; D008958:Models, Molecular; D011228:Prealbumin / Q000737:chemistry* / Q000235:genetics; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics
D001704:Biopolymers; D011228:Prealbumin; D011994:Recombinant Proteins