Designing transthyretin mutants affecting tetrameric structure: implications in amyloidogenicity

Citation
C. Redondo et al., Designing transthyretin mutants affecting tetrameric structure: implications in amyloidogenicity, BIOCHEM J, 348, 2000, pp. 167-172
Citations number
20
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Biochemistry & Biophysics
Journal title
BIOCHEMICAL JOURNAL
ISSN journal
0264-6021 → ACNP
Volume
348
Year of publication
2000
Part
1
Pages
167 - 172
Database
ISI
SICI code
0264-6021(20000515)348:<167:DTMATS>2.0.ZU;2-N
Abstract
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 structu ral studies. We have designed four mutated proteins with the purpose of sta bilizing [Ser(117) --> Cys (S117C) and Glu(92) --> Cys (E92C)] or destabili zing [Asp(18) --> Asn (D18N) and Leu(110) --> Ala (D110A)] the dimer/tetram er interactions in TTR, aiming at elucidating structural determinants in am yloidogenesis. The resistance of the mutated proteins to dissociation was a nalysed by HPLC studies of diluted TTR preparations. Both 'stabilized' muta nts migrated as tetramers and, upon dilution, no other TTR species was obse rved, confirming the increased resistance to dissociation. For the 'destabi lized' mutants, a mixture of tetrameric and monomeric forms co-existed at l ow dilution and the latter increased upon 10-fold dilution. Both of the des tabilizing mutants formed amyloid in vitro when acidified. This result indi cated that both the AB loop of TTR, destabilized in D18N, and the hydrophob ic interactions affecting the dimer-dimer interfaces in L110A are implicate d in the stability of the tetrameric structure. The stabilized mutants, whi ch were dimeric in nature through disulphide bonding, were unable to polyme rize into amyloid, even at pH 3.2. When the amyloid formation assay was rep eated in the presence of 2-mercaptoethanol, upon disruption of the S-S brid ges of these stable dimers, amyloid fibril formation was observed. This exp erimental evidence suggests that monomers, rather than dimers, are the repe ating structural subunit comprising the amyloid fibrils.