Abstract

Familial Alzheimer disease-causing mutations in the presenilins increase production of longer pathogenic amyloid beta-peptides (A beta(42/43)) by altering gamma-secretase activity. The mechanism underlying this effect remains unknown, although it has been proposed that heteromeric macromolecular complexes containing presenilins mediate gamma-secretase cleavage of the amyloid beta-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 A beta43, but did not support physiological PS1 endoproteolysis or A beta40 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 A beta43 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 gamma-secretase and that the effects of PS1 mutations and gamma-secretase inhibitors on the specificity are mediated through a common mechanism.

Highlights

  • The amyloid ␤-precursor protein (APP),1 Notch, ErbB-4, and several other unrelated type 1 transmembrane (TM) proteins undergo ␥-secretase cleavage within their TM domains [1, 2]

  • 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 amyloid ␤-peptide (A␤)43, but did not support physiological PS1 endoproteolysis or A␤40 generation

  • Identification of Novel Endoproteolysis-impaired PS1 Mutants by Random Mutagenesis Screening—We generated and screened a randomly mutagenized PS1 cDNA library to identify mutants that were defective in PS1 endoproteolysis

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Summary

Introduction

The amyloid ␤-precursor protein (APP),1 Notch, ErbB-4, and several other unrelated type 1 transmembrane (TM) proteins undergo ␥-secretase cleavage within their TM domains [1, 2]. Evaluation of PS-dependent Cleavages of APP and Notch—For analysis of AICD generation, crude membrane fractions were obtained from HEK293 cells expressing various PS1 mutants and solubilized in lysis buffer containing 0.5% CHAPSO and 5 mM 1,10-phenanthroline (Sigma). These mutants exhibited dominant phenotypes for the secretion of A␤40 and A␤42/43 presumably through the replacement of endogenous PSs. To assess the ␥-secretase activity of these mutants in the absence of endogenous PSs, we transfected each of these PS1 mutants together with human APP into PS-null MEFs using retroviral vectors and measured A␤ levels in the conditioned media from these cells (Fig. 3A).

Results
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