Figure 1. FAD PSEN1 mutations inhibit processive proteolysis of C99 by γ-Secretase.

(A) Schematic of proteolytic processing of APP substrate by γ-Secretase.

(B) Quantitative western blotting of total AICD, using purified substrate C100-FLAG to generate a standard curve for densitometry.

(C) MALDI-TOF analysis shows ratios of peak heights of AICD49–99/AICD50–99 produced by γ-Secretase with WT versus six FAD mutants of PSEN1.

(D) Quantification of AICD49–99 and AICD50–99 production, corresponding to Aβ48 and Aβ49 production, respectively.

(E) Schematic of light-versus heavy-isotope labeling of APP substrate for LC-MS/MS analysis of effects of PSEN1 FAD mutations on carboxypeptidase trimming steps.

(F) Bar graphs of small peptide coproduct for each trimming step. Blue graphs, first, second, and third trimming step for the Aβ49 → Aβ40 pathway; red graphs, trimming steps for the Aβ48 → Aβ38 pathway; dark and light bars, coproduct formation from WT and FAD-mutant γ-Secretase, respectively.

(G) Bar graphs of percentage cleavage efficiency of each trimming step relative to WT enzyme. Where the level of precursor Ab peptide for a given trimming step is zero, the efficiency of this cleavage event could not be determined (nd). In all graphs, n = 3, unpaired two-tailed t test compared FAD mutant with WT, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001).