Two-Hybrid System as a Model to Study the Interaction of β -Amyloid Peptide Monomers

The kinetics of amyloid fibril formation by β -amyloid peptide (Aβ ) are typical of a nucleation-dependent polymerization mechanism. This type of mechanism suggests that the study of the interaction of Aβ with itself can provide some valuable insights into Alzheimer disease amyloidosis. Interaction of Aβ with itself was explored with the yeast two-hybrid system. Fusion proteins were created by linking the Aβ 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 (β -galactosidase) and LEU2 (leucine utilization) genes under the control of LexA-dependent operators. This approach suggests that the Aβ 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 Aβ , indicating that the observed Aβ -Aβ interaction was specific. Specificity was further shown by the finding that no significant interaction was observed in yeast expressing LexA-Aβ bait when the B42 transactivation domain was fused to an Aβ fragment with Phe-Phe at residues 19 and 20 replaced by Thr-Thr (Aβ 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 Aβ -(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 Aβ monomers and to define the peptide sequences that may be important in nucleation-dependent aggregation.