Random Mutagenesis of Presenilin-1 Identifies Novel Mutants Exclusively Generating Long Amyloid β-Peptides

Familial Alzheimer disease-causing mutations in the presenilins increase production of longer pathogenic amyloid β-peptides (Aβ42/43) by altering γ-secretase activity. The mechanism underlying this effect remains unknown, although it has been proposed that heteromeric macromolecular complexes containing presenilins mediate γ-secretase cleavage of the amyloid β-precursor protein. Using a random mutagenesis screen of presenilin-1 (PS1) for PS1 endoproteolysis-impairing mutations, we identified five unique mutants, including R278I-PS1 and L435H-PS1, that exclusively generated a high level of Aβ43, but did not support physiological PS1 endoproteolysis or Aβ40 generation. These mutants did not measurably alter the molecular size or subcellular localization of PS1 complexes. Pharmacological studies indicated that the up-regulation of activity for Aβ43 generation by these mutations was not further enhanced by the difluoroketone inhibitor DFK167 and was refractory to inhibition by sulindac sulfide. These results suggest that PS1 mutations can lead to a wide spectrum of changes in the activity and specificity of γ-secretase and that the effects of PS1 mutations and γ-secretase inhibitors on the specificity are mediated through a common mechanism