Two-hybrid system as a model to study the interaction of beta-amyloid peptide monomers

The kinetics of amyloid fibril formation by beta-amyloid peptide (A beta) are typical of a nucleation-dependent polymerization mechanism, This type of mechanism suggests that the study of the interaction of A beta with itself can provide some valuable insights into Alzheimer disease amyloidosis. Interaction of A beta with itself was explored with the yeast two-hybrid system, Fusion proteins were created by linking the A beta fragment to a LexA DNA-binding domain (bait) and also to a B42 transactivation domain (prey), Protein-protein interactions were measured by expression of these fusion proteins in Saccharomyces cerevisiae harboring lacZ (beta-galactosidase) and LEU2 (leucine utilization) genes under the control of LexA-dependent operators, This approach suggests that the A beta molecule is capable of interacting with itself in vivo in the yeast cell nucleus. LexA protein fused to the Drosophila protein bicoid (LexA-bicoid) failed to interact with the B42 fragment fused to A beta, indicating that the observed A beta-A beta interaction was specific, Specificity was further shown by the finding that no significant interaction was observed in yeast expressing LexA-A beta bait when the B42 transactivation domain was fused to an A beta fragment with Phe-Phe at residues 19 and 20 replaced by Thr-Thr (A beta TT), a finding that is consistent with in vitro observations made by others, Moreover, when a peptide fragment bearing this substitution was mixed with native A beta-(1-40), it inhibited formation of fibrils in vitro as examined by electron microscopy, The findings presented in this paper suggest that the two-hybrid system can be used to study the interaction of A beta monomers and to define the peptide sequences that may be important in nucleation-dependent aggregation.