Mutations that reduce aggregation of the Alzheimer's Aβ42 peptide:: an unbiased search for the sequence determinants of Aβ amyloidogenesis

The primary component of amyloid plaque in the brains of Alzheimer's patients is the 42 residue amyloid-p-peptide (Abeta42). Although the amino acid residue sequence of Abeta42 is known, the molecular determinants of Abeta amyloiclogenesis have not been elucidated. To facilitate an unbiased search for the sequence determinants of AP aggregation, we developed a genetic screen that couples a readily observable phenotype in E. coli to the ability of a mutation in Abeta42 to reduce aggregation. The screen is based on our finding that fusions of the wild-type Abeta42 sequence to green fluorescent protein (GFP) form insoluble aggregates in which GFP is inactive. Cells expressing such fusions do not fluoresce. To isolate variants of Abeta42 with reduced tendencies to aggregate, we constructed and screened libraries of Abeta42-GFP fusions in which the sequence of Abeta42 was mutated randomly. Cells expressing GFP fusions to soluble (non-aggregating) variants of Abeta42 exhibit green fluorescence. Implementation of this screen enabled the isolation of 36 variants of Abeta42 with reduced tendencies to aggregate. The sequences of most of these variants are consistent with previous models implicating hydrophobic regions as determinants of Abeta42 aggregation. Some of the variants, however, contain amino acid substitutions not implicated in pre-existing models of Abeta amyloiclogenesis. (C) 2002 Elsevier Science Ltd. All rights reserved.