title: Two-hybrid system as a model to study the interaction of beta-amyloid peptide monomers.
abstract: The kinetics of amyloid fibril formation by beta-amyloid peptide (Abeta) are typical of a nucleation-dependent polymerization mechanism. This type of mechanism suggests that the study of the interaction of Abeta with itself can provide some valuable insights into Alzheimer disease amyloidosis. Interaction of Abeta with itself was explored with the yeast two-hybrid system. Fusion proteins were created by linking the Abeta 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 Abeta 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 Abeta, indicating that the observed Abeta-Abeta interaction was specific. Specificity was further shown by the finding that no significant interaction was observed in yeast expressing LexA-Abeta bait when the B42 transactivation domain was fused to an Abeta fragment with Phe-Phe at residues 19 and 20 replaced by Thr-Thr (AbetaTT), a finding that is consistent with in vitro observations made by others. Moreover, when a peptide fragment bearing this substitution was mixed with native Abeta-(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 Abeta monomers and to define the peptide sequences that may be important in nucleation-dependent aggregation.
authors.affiliation: Neuroscience Therapeutic Domain, Hoechst Marion Roussel Inc., Somerville, NJ 08876, USA.
authors.forename: S R
authors.initials: SR
authors.lastname: Hughes
authors.forename: S
authors.initials: S
authors.lastname: Goyal
authors.forename: J E
authors.initials: JE
authors.lastname: Sun
authors.forename: P
authors.initials: P
authors.lastname: Gonzalez-DeWhitt
authors.forename: M A
authors.initials: MA
authors.lastname: Fortes
authors.forename: N G
authors.initials: NG
authors.lastname: Riedel
authors.forename: S R
authors.initials: SR
authors.lastname: Sahasrabudhe
mesh_terms: D000544:Alzheimer Disease; D016229:Amyloid beta-Peptides / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism; D001483:Base Sequence; D017931:DNA Primers / Q000737:chemistry; D029721:Drosophila Proteins; D004926:Escherichia coli; D018398:Homeodomain Proteins; D006801:Humans; D007301:Insect Hormones / Q000378:metabolism; D046911:Macromolecular Substances; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins; D012441:Saccharomyces cerevisiae; D012697:Serine Endopeptidases / Q000378:metabolism; D015534:Trans-Activators; D000099160:LexA Repressor Protein
chemical_list: D016229:Amyloid beta-Peptides; D001426:Bacterial Proteins; D017931:DNA Primers; D029721:Drosophila Proteins; D018398:Homeodomain Proteins; D007301:Insect Hormones; D046911:Macromolecular Substances; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D012697:Serine Endopeptidases; D015534:Trans-Activators; D000099160:LexA Repressor Protein; C056478:bcd protein, Drosophila