Partial purification and characterization of gamma-secretase from post-mortem human brain.
One characteristic feature of Alzheimer's disease is the deposition of amyloid beta-peptide (Abeta) as amyloid plaques within specific regions of the human brain. Abeta is derived from the amyloid beta-peptide precursor protein (beta-APP) by the intramembranous cleavage activity of gamma-secretase. Studies in cells have revealed that gamma-secretase is a large multimeric membrane-bound protein complex that is functionally dependent on several proteins, including presenilin, nicastrin, Aph-1, and Pen-2. However, the precise biochemical and molecular nature of gamma-secretase is as yet to be fully elucidated, and no investigations have analyzed gamma-secretase in human brain. To address this we have developed a novel in vitro gamma-secretase activity assay using detergent-solubilized cell membranes and a beta-APP-derived fluorescent probe. We report that human brain-derived gamma-secretase activity co-purifies with a high molecular weight protein complex comprising presenilin, nicastrin, Aph-1, and Pen-2. The inhibitor profile and solubility characteristics of brain-derived gamma-secretase are similar to those described in cells, and proteolysis occurs at the Abeta40- and Abeta42-generating cleavage sites. The ability to isolate gamma-secretase from post-mortem human brain may facilitate the identification of brain-specific modulators of beta-APP processing and provide new insights into the biology of this important factor in the pathogenesis of Alzheimer's disease.
Farmery
Mark R
MR
Karolinska Institutet and Sumitomo Pharmaceuticals Alzheimer Center, Neurotec, Novum, Huddinge, SE-141 57 Sweden. mark.farmery@neurotec.ki.se
Tjernberg
Lars O
LO
Pursglove
Sharon E
SE
Bergman
Anna
A
Winblad
Bengt
B
Näslund
Jan
J
D000544:Alzheimer Disease / Q000201:enzymology; D053829:Amyloid Precursor Protein Secretases; D016229:Amyloid beta-Peptides / Q000032:analysis* / Q000378:metabolism; D016282:Aspartic Acid Endopeptidases; D001681:Biological Assay; D001921:Brain / Q000201:enzymology*; D002102:Cadaver; D010450:Endopeptidases / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism; D005456:Fluorescent Dyes; D006801:Humans; D066298:In Vitro Techniques
D016229:Amyloid beta-Peptides; D005456:Fluorescent Dyes; D053829:Amyloid Precursor Protein Secretases; D010450:Endopeptidases; D016282:Aspartic Acid Endopeptidases; C509280:BACE1 protein, human