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O1–08–03: Failure in beta‐amyloid degradation in the endosomal/lysosomal pathway results in intracellular beta‐amyloid accumulation within lipid rafts
Author(s) -
PachecoQuinto Javier,
Eckman Cari,
Horowitz Julia,
Kennedy Sami,
McCann Mondona,
Woods Chelsie,
Eckman Elizabeth
Publication year - 2013
Publication title -
alzheimer's and dementia
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 6.713
H-Index - 118
eISSN - 1552-5279
pISSN - 1552-5260
DOI - 10.1016/j.jalz.2013.04.094
Subject(s) - intracellular , endosome , extracellular , microbiology and biotechnology , brefeldin a , biochemistry , biology , chemistry , golgi apparatus , endoplasmic reticulum
that range from 38 to 49 residues. Evidence in cells suggests that the protease first cleaves the 99-residue APP-derived substrate (C99) within the transmembrane domain at the so-called e site to release the intracellular domain and produce a 48or 49-residue A b. Subsequent cleavage roughly every three residues from the carboxy terminus is thought to lead to two pathways toward shorter secreted forms of Ab: Ab 49-Ab 46-Ab 43-Ab 40 and Ab 48-Ab 45-Ab 42-Ab 38. We sought to validate the carboxypeptidase function of gamma -secretase and to test the existence of these two pathways and extent of crossover between them in a purified and reconstituted system.Methods: Human gamma -secretase complexes were purified from Chinese hamster ovary cells over-expressing all four components: presenilin-1, nicastrin, Aph-1 and Pen-2, with the purified enzyme containing added lipids and solubilized in CHAPSO detergent. Purified recombinant Met-C99-Flag substrate (C100Flag) or synthetic Ab 48 or Ab 49 was added to 1-2 mM final concentration. After 4 h incubation, Ab 40 and Ab 42 were each quantified by specific ELISAs. In other experiments, CHAPSOwas removed from the purified enzyme preparations using styrene beads to allow reconstitution of the protease complex into lipid vesicles prior to substrate addition. Results: Addition of Ab 48 or Ab49 directly into purified preparations of gamma -secretase led to the formation of Ab 40 and Ab 42, whether the protease complex was detergent-solubilized or reconstituted into lipid vesicles. For Ab 48, the ratio of Ab 42 to Ab 40 was 8:1, while for Ab 49, the ratio was 1:8. Production of Ab 40 and Ab 42 was prevented by concomitant addition of gamma -secretase inhibitor, prior heat inactivation of the enzyme preparation, or use of gamma -secretase complexes with mutation of the catalytic aspartates of presenilin-1. Conclusions: The carboxypeptidase activity that trims long Ab peptides to shorter ones is an intrinsic property of the protease complex. The ratio of Ab 42 to Ab 40 produced from Ab 48 or Ab 49 is not determined by incorporation into membranes. These results are consistent with the dual pathways outlined above, although a small degree of crossover between these pathways can apparently occur.