Substrate recruitment of γ‐secretase and mechanism of clinical presenilin mutations revealed by photoaffinity mapping

Abstract

Intramembrane proteases execute fundamental biological processes ranging from crucial signaling events to general membrane proteostasis. Despite the availability of structural information on these proteases, it remains unclear how these enzymes bind and recruit substrates, particularly for the Alzheimer's diseaseassociated c-secretase. Systematically scanning amyloid precursor protein substrates containing a genetically inserted photocrosslinkable amino acid for binding to c-secretase allowed us to identify residues contacting the protease. These were primarily found in the transmembrane cleavage domain of the substrate and were also present in the extramembranous domains. The N-terminal fragment of the catalytic subunit presenilin was determined as principal substrate-binding site. Clinical presenilin mutations altered substrate binding in the active site region, implying a pathogenic mechanism for familial Alzheimer's disease. Remarkably, PEN-2 was identified besides nicastrin as additional substrate-binding subunit. Probing proteolysis of crosslinked substrates revealed a mechanistic model of how these subunits interact to mediate a stepwise transfer of bound substrate to the catalytic site. We propose that sequential binding steps might be common for intramembrane proteases to sample and select cognate substrates for catalysis.

High-efficient production and biophysical characterisation of nicastrin and its interaction with APPC100

“…But the low-μM K D value obtained here is consistent with the initial-docking-site-model for the recognition of substrate by γ-secretase, especially if additional interactions between substrate and PEN-243 in the complex are considered.…”

Toward the Characterization of DAPT Interactions with γ‐Secretase

“…[29] Those results and further photolabeling experiments suggested that DAPT inhibition is distinct from TSA, but both compete for the active site and exhibit partially overlapped binding pockets. [26] Similar behavior was found for the GSI avagacestat (BMS-708163), which binds within TM2, TM3, TM5, and TM7 of PS1 component without interacting with the catalytic dyad. [26] Similar behavior was found for the GSI avagacestat (BMS-708163), which binds within TM2, TM3, TM5, and TM7 of PS1 component without interacting with the catalytic dyad.…”

“…[22] Based on their effect on GS activity,t hese molecules have been classified in GS inhibitors (GSIs) andG Sm odulators (GSMs). [24][25][26] Ar ecent cryo-electron microscopy (cryo-EM) structure of GS was solved with the potent non-transition-stated ipeptidebased inhibitor DAPT at the PS1 active site ( Figure 1B). On the other hand, GSMs intend to modifyt he cleavage pattern of C99p eptidet od ecrease Ab42 secretion withouta ltering the trimming of other substrates (e.g.,N otch-1).…”

“…However,D APT displacementt owardt he bilayer center (x = 0.0 nm) resulted in af ree-energy increment due to the high mobility of the lipid tails in the transmembrane region. [26] PMF profiles obtained from pulling DAPT from the PS1 active site through both entries suggest that the ligand is more likely to access the site through TM2 and TM3 (ES1, Figure 2B and Figure S4). The PMF profile and our MD simulations demonstrate that DAPT has ah ighp robability of adhering to the membrane surface, which could facilitatei ts recognition by the membrane-buried PS1 active site.…”

Transmembrane Substrate Determinants for γ-Secretase Processing of APP CTFβ

“…20, 38 In addition, a recent study demonstrated that APP CTFβ residues A42, V44, I45, L49, M51, and L52 directly interact with the C-terminal or N-terminal fragments of PS1 62 . These results are consistent with our observations that deleting substrate residues around the ε sites significantly reduces ε site cleavage, as interactions between PS1 and CTFβ at positions L49, M51, and L52 would be lost.…”