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 gamma-secretase complex mediates the final proteolytic event in Alzheimer's disease amyloid-beta 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 gamma-sites to form C-terminally heterogeneous amyloid-beta and cleaves at an epsilon-site to release the intracellular domain or epsilon-C-terminal fragment. In this work, two novel in vitro gamma-secretase assays are developed to further explore the biochemical characteristics of gamma-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-beta and an epsilon-C-terminal fragment were observed. Upon purification this substrate was used in parallel with a transfected source of substrate to measure gamma-secretase activity from detergent extracted membranes. With these systems, it was determined that recovery of size-fractionated cellular and tissue-derived gamma-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 epsilon-C-terminal fragments can be elevated. Lastly, we show that zinc causes an increase in the apparent molecular mass of an amyloid precursor protein gamma-secretase substrate and inhibits its cleavage. These studies further refine our knowledge of the complexes and biochemical factors needed for gamma-secretase activity and suggest a mechanism by which zinc dysregulation may contribute to Alzheimer's disease pathogenesis.
Hoke
David E
DE
Department of Pathology, The University of Melbourne and the Mental Health Research Institute, Parkville, Victoria, Australia. david.hoke@med.monash.edu.au
Tan
Jiang-Li
JL
Ilaya
Nancy T
NT
Culvenor
Janetta G
JG
Smith
Stephanie J
SJ
White
Anthony R
AR
Masters
Colin L
CL
Evin
Geneviève M
GM
D053829:Amyloid Precursor Protein Secretases; D016564:Amyloid beta-Protein Precursor / Q000378:metabolism*; D000818:Animals; D001921:Brain / Q000378:metabolism; D019556:COS Cells; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism; D002522:Chlorocebus aethiops; D002850:Chromatography, Gel; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D010450:Endopeptidases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006168:Guinea Pigs; D008565:Membrane Proteins / Q000378:metabolism*; D008566:Membranes / Q000378:metabolism; D008970:Molecular Weight; D046912:Multiprotein Complexes / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D011499:Protein Processing, Post-Translational / Q000187:drug effects*; D013379:Substrate Specificity; D015032:Zinc / Q000494:pharmacology*
D016564:Amyloid beta-Protein Precursor; D008565:Membrane Proteins; D046912:Multiprotein Complexes; D053829:Amyloid Precursor Protein Secretases; D010450:Endopeptidases; D015032:Zinc; D004121:Dimethyl Sulfoxide