In vitro gamma‐secretase cleavage of the Alzheimer's amyloid precursor protein correlates to a subset of presenilin complexes and is inhibited by zinc

The γ-secretase complex mediates the final proteolytic event in Alzheimer's disease amyloid-β biogenesis. This membrane complex of presenilin, anterior pharynx defective, nicastrin, and presenilin enhancer-2 cleaves the C-terminal 99-amino acid fragment of the amyloid precursor protein intramembranously at γ-sites to form C-terminally heterogeneous amyloid-β and cleaves at an e-site to release the intracellular domain or e-C-terminal fragment. In this work, two novel in vitroγ-secretase assays are developed to further explore the biochemical characteristics of γ-secretase activity. During development of a bacterial expression system for a substrate based on the amyloid precursor protein C-terminal 99-amino acid sequence, fragments similar to amyloid-β and an e-C-terminal fragment were observed. Upon purification this substrate was used in parallel with a transfected source of substrate to measure γ-secretase activity from detergent extracted membranes. With these systems, it was determined that recovery of size-fractionated cellular and tissue-derived γ-secretase activity is dependent upon detergent concentration and that activity correlates to a subset of high molecular mass presenilin complexes. We also show that by changing the solvent environment with dimethyl sulfoxide, detection of e-C-terminal fragments can be elevated. Lastly, we show that zinc causes an increase in the apparent molecular mass of an amyloid precursor protein γ-secretase substrate and inhibits its cleavage. These studies further refine our knowledge of the complexes and biochemical factors needed for γ-secretase activity and suggest a mechanism by which zinc dysregulation may contribute to Alzheimer's disease pathogenesis.