Mutations that reduce aggregation of the Alzheimer's Abeta42 peptide: an unbiased search for the sequence determinants of Abeta amyloidogenesis.
The primary component of amyloid plaque in the brains of Alzheimer's patients is the 42 residue amyloid-beta-peptide (Abeta42). Although the amino acid residue sequence of Abeta42 is known, the molecular determinants of Abeta amyloidogenesis have not been elucidated. To facilitate an unbiased search for the sequence determinants of Abeta 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 amyloidogenesis.
Wurth
Christine
C
Department of Chemistry, Princeton University, NJ 08544, USA.
Guimard
Nathalie K
NK
Hecht
Michael H
MH
D000544:Alzheimer Disease / Q000235:genetics* / Q000378:metabolism*; D016229:Amyloid beta-Peptides / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000686:Amyloidosis / Q000235:genetics* / Q000378:metabolism*; D015982:Bias; D001665:Binding Sites; D002942:Circular Dichroism; D003224:Congo Red; D005453:Fluorescence; D049452:Green Fluorescent Proteins; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D009154:Mutation / Q000235:genetics*; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011485:Protein Binding; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012995:Solubility
D016229:Amyloid beta-Peptides; D008164:Luminescent Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; C075222:amyloid beta-protein (1-42); D049452:Green Fluorescent Proteins; D003224:Congo Red