Mechanism of γ-secretase cleavage activation: Is γ-secretase regulated through autoinhibition involving the presenilin-1 exon 9 loop?

Maturation of γ-secretase requires an endoproteolytic cleavage in presenilin-1 (PS1) within a peptide loop encoded by exon 9 of the corresponding gene. Deletion of the loop has been demonstrated to cause familial Alzheimer's disease. A synthetic peptide corresponding to the loop sequence was found to inhibit γ-secretase in a cell-free enzymatic assay with an IC50 of 2.1 μM, a value similar to the Km (3.5 μM) for the substrate C100. Truncation at either end, single amino acid substitutions at certain residues, sequence reversal, or randomization reduced its potency. Similar results were also observed in a cell-based assay using HEK293 cells expressing APP. In contrast to small-molecule γ-secretase inhibitors, kinetic inhibition studies demonstrated competitive inhibition of γ-secretase by the exon 9 peptide. Consistent with this finding, inhibitor cross-competition kinetics indicated noncompetitive binding between the exon 9 peptide and L685458, a transition-state analogue presumably binding at the catalyt...