Familial Alzheimer’s disease (FAD) is caused by dominant mutations in the amyloid-ß (Aß) precursor
protein (APP) and presenilin-1 and -2 (PSEN1, PSEN2). APP is cleaved by ß-secretase, then within its single
transmembrane domain (TMD) by ¿-secretase to produce Aß, which deposits as cerebral plaques. PSEN is the
catalytic component of ¿-secretase, a membrane-embedded protease complex. Thus, FAD mutations are only
in the substrate and protease that produce Aß; nevertheless, pathogenic mechanisms remain controversial.
Most PSEN FAD mutations show reduced proteolysis (loss of function) but also increase proportions of
aggregation-prone 42-residue Aß peptide (Aß42) (gain of function). However, ¿-secretase has multiple
proteolytic functions: Initial endoproteolytic (e) cleavage of APP substrate produces long Aß that is trimmed via
a carboxypeptidase activity, and FAD-mutant ¿-secretases are deficient in this trimming function.
New understanding of multiple proteolytic functions of ¿-secretase along with recent cryo-EM structure
elucidation of the protease-substrate complex now make possible a deeper understanding of effects of FAD
mutations. The goal here is to combine chemical, structural, and computational biology to elucidate how
presenilin FAD mutations alter ¿-secretase structure, dynamics, and function. Such understanding should give
insight into how this membrane-embedded protease complex recognizes and processively proteolyzes
transmembrane substrates, provide critical clues to pathogenic mechanisms of FAD, and suggest new
strategies for prevention of FAD. To this end, we propose to:
(1) Develop chemical probes to trap ¿-secretase in different stages of substrate interaction for structural
analysis by cryo-EM. We developed full TMD substrate mimics to trap active enzyme in a conformation poised
for intramembrane proteolysis. Designed variations should allow visualization of the transition states for e
proteolysis, carboxypeptidase cleavage, TMD helix unwinding, and lateral gating of substrate.
(2) Perform molecular dynamics (MD) simulations of substrate interaction with FAD-mutant ¿-secretase.
We computationally restored catalytic aspartates, modeled entry of water to the active site, and captured
activation of the computationally restored WT enzyme. We will determine effects of FAD PSEN1 mutations on
¿-secretase structural dynamics and interaction with APP substrate and TMD mimics.
(3) Develop synthetic substrate probes for analysis of proteolytic dysfunction of FAD-mutant ¿-secretase.
We developed a set of such functional probes of ¿-secretase processing of APP TMD, validating them as
convenient and appropriate synthetic surrogates for APP substrate. We will employ these and other proposed
substrate probes to determine effects of FAD-mutant ¿-secretases on e proteolysis and specific
carboxypeptidase trimming steps.