γ-Secretase, Evidence for Multiple Proteolytic Activities and Influence of Membrane Positioning of Substrate on Generation of Amyloid β Peptides of Varying Length

Michael Paul Murphy,Lesley Jill Hickman,Christopher Benjamin Eckman,Sacha Noelle Uljon,Rong Wang,Todd Eliot Golde

doi:10.1074/jbc.274.17.11914

Michael Paul Murphy, Lesley Jill Hickman + Show 4 more

Open Access

https://doi.org/10.1074/jbc.274.17.11914

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Abstract

gamma-Secretase activity is the final cleavage event that releases the amyloid beta peptide (Abeta) from the beta-secretase cleaved carboxyl-terminal fragment of the amyloid beta protein precursor (APP). No protease responsible for this highly unusual, purportedly intramembranous, cleavage has been definitively identified. We examined the substrate specificity of gamma-secretase by mutating various residues within or adjacent to the transmembrane domain of the APP and then analyzing Abeta production from cells transfected with these mutant APPs by enzyme-linked immunosorbent assay and mass spectrometry. Abeta production was also analyzed from a subset of transmembrane domain APP mutants that showed dramatic shifts in gamma-secretase cleavage in the presence or absence of pepstatin, an inhibitor of gamma-secretase activity. These studies demonstrate that gamma-secretase's cleavage specificity is primarily determined by location of the gamma-secretase cleavage site of APP with respect to the membrane, and that gamma-secretase activity is due to the action of multiple proteases exhibiting both a pepstatin- sensitive activity and a pepstatin-insensitive activity. Given that gamma-secretase is a major therapeutic target in Alzheimer's disease these studies provide important information with respect to the mechanism of Abeta production that will direct efforts to isolate the gamma-secretases and potentially to develop effective therapeutic inhibitors of pathologically relevant gamma-secretase activities.

Highlights

The 4-kDa amyloid ␤ protein (A␤)1 that is invariably deposited as amyloid in Alzheimer’s disease (AD) is a normally secreted proteolytic product of the amyloid ␤ protein precursor (APP) [1,2,3]
We examined the substrate specificity of ␥-secretase by mutating various residues within or adjacent to the transmembrane domain of the APP and analyzing A␤ production from cells transfected with these mutant APPs by enzyme-linked immunosorbent assay and mass spectrometry
1 The abbreviations used are: A␤, amyloid ␤ protein; APP, amyloid ␤ protein precursor; AD, Alzheimer’s disease; FAD, familial Alzheimer’s disease; NL, APPK670N,M671L mutation; TMD, transmembrane domain; PS, presenilin; Me2SO, dimethyl sulfoxide; KA␤28, lysine at A␤28; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline; PBSB, phosphatebuffered saline with bovine serum albumin; flAPP, full-length APP; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine

Summary

The abbreviations used are

A␤, amyloid ␤ protein; APP, amyloid ␤ protein precursor; AD, Alzheimer’s disease; FAD, familial Alzheimer’s disease; NL, APPK670N,M671L mutation (numbering based on APP770 isoform); TMD, transmembrane domain; PS, presenilin; Me2SO, dimethyl sulfoxide; KA␤28, lysine at A␤28; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline; PBSB, phosphatebuffered saline with bovine serum albumin; flAPP, full-length APP; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. While previous studies have shown that ␤-secretase cleavage, like most proteolytic cleavages, exhibits fairly rigid primary amino acid sequence requirements [15], similar studies of the more complex ␥-secretase activity demonstrates fairly loose specificity [16, 17]. These studies, which either examine effects on total A␤ production [16] or effects of mutations at a single residue, A␤43 [17], provide no compelling mechanisms for the observed alterations in cleavage associated with FAD-linked mutations. All mutations were made in a background of the APP695NL mutation in order to increase absolute amounts of A␤ peptides generated without affecting ␥-cleavage as this mutation increases activity at the ␤-secretase site without altering the relative amounts of either A␤40 or A␤42 [5, 23,24,25]

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION