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-β-peptide (Aβ42). Although the amino acid residue sequence of Aβ42 is known the molecular determinants of Aβ amyloidogenesis have not been elucidated. To facilitate an unbiased search for the sequence determinants of Aβ aggregation we developed a genetic screen that couples a readily observable phenotype in E. coli to the ability of a mutation in Aβ42 to reduce aggregation. The screen is based on our finding that fusions of the wild-type Aβ42 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 Aβ42 with reduced tendencies to aggregate we constructed and screened libraries of Aβ42-GFP fusions in which the sequence of Aβ42 was mutated randomly. Cells expressing GFP fusions to soluble (non-aggregating) variants of Aβ42 exhibit green fluorescence. Implementation of this screen enabled the isolation of 36 variants of Aβ42 with reduced tendencies to aggregate. The sequences of most of these variants are consistent with previous models implicating hydrophobic regions as determinants of Aβ42 aggregation. Some of the variants however contain amino acid substitutions not implicated in pre-existing models of Aβ amyloidogenesis.