Abstract
γ-Secretase is responsible for the proteolysis of amyloid precursor protein (APP) into amyloid-beta (Aβ) peptides, which are centrally implicated in the pathogenesis of Alzheimer’s disease (AD). The biochemical mechanism of how processing by γ-secretase is regulated, especially as regards the interaction between enzyme and substrate, remains largely unknown. Here, mutagenesis reveals that the hydrophilic loop-1 (HL-1) of presenilin-1 (PS1) is critical for both γ-secretase step-wise cleavages (processivity) and its allosteric modulation by heterocyclic γ-modulatory compounds. Systematic mutagenesis of HL-1, including all of its familial AD mutations and additional engineered variants, and quantification of the resultant Aβ products show that HL-1 is necessary for proper sequential γ-secretase processivity. We identify Y106, L113, and Y115 in HL-1 as key targets for heterocyclic γ-secretase modulators (GSMs) to stimulate processing of pathogenic Aβ peptides. Further, we confirm that the GxxxG domain in the APP transmembrane region functions as a critical substrate motif for γ-secretase processivity: a G29A substitution in APP-C99 mimics the beneficial effects of GSMs. Together, these findings provide a molecular basis for the structural regulation of γ-processivity by enzyme and substrate, facilitating the rational design of new GSMs that lower AD-initiating amyloidogenic Aβ peptides.
Highlights
The generation of amyloid β-peptides (Aβ) via sequential cleavages of amyloid precursor protein (APP) by β-secretase (BACE1) and the presenilin/γsecretase complex is central to the initiation of Alzheimer’s disease (AD) [1,2,3]. γ-Secretase is a high-molecular-weight (HMW) multiprotein complex with 20 transmembrane domains (TMDs) and has an unusual intramembrane di-aspartyl catalytic site within the presenilin component
We generated PS1 constructs for all 17 known familial Alzheimer’s disease (FAD) mutations within the hydrophilic loop1 (HL-1) region and transiently coexpressed them in the HEK293 PS dKO line together with wt human APP
Quantification of the secreted Aβ species in conditioned media (CM) showed that compared with wt-PS1, 1) 15 of 16 FAD mutations decreased total secreted Aβs; 2) all 16 mutations increased the Aβ42/40 ratio in the CM, as expected; and 3) all 16 mutations decreased Aβ37/40, Aβ37/ 42, and Aβ38/42 ratios (Fig. 1C)
Summary
The generation of amyloid β-peptides (Aβ) via sequential cleavages of APP by β-secretase (BACE1) and the presenilin/γsecretase complex is central to the initiation of Alzheimer’s disease (AD) [1,2,3]. γ-Secretase is a high-molecular-weight (HMW) multiprotein complex with 20 transmembrane domains (TMDs) and has an unusual intramembrane di-aspartyl catalytic site within the presenilin component. A detailed biochemical study of this HL-1 region is essential to further our understanding of native γ-secretase enzymatic function To this end, we have analyzed all individual residues (aa 101–120) of HL-1 of PS1 with regard to 1) the enzymatic activity of the FAD mutations therein as well as the effects of alanine scanning mutagenesis (Ala substitutions at each residue within this region); 2) the performance of five γ-secretase modulators (GSMs) of two general classes on Aβ generation from the HL-1 FAD-mutant residues; and 3) the response of these GSMs to the Ala substitutions. We explored the mechanism of GSMs by examining the function of the GxxxG domain in the APP transmembrane region and found that G29 (Aβ numbering) is a key residue for proper γ-secretase processivity.
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