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 ε‐site to release the intracellular domain or ε‐C‐terminal fragment. In this work, two novel in vitro γ‐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 ε‐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 ε‐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.