Presenilin 1 is linked with gamma-secretase activity in the detergent solubilized state.
gamma-Secretase is a membrane-associated protease that cleaves within the transmembrane region of amyloid precursor protein to generate the C termini of the two Abeta peptide isoforms, Abeta40 and Abeta42. Here we report the detergent solubilization and partial characterization of gamma-secretase. The activity of solubilized gamma-secretase was measured with a recombinant substrate, C100Flag, consisting largely of the C-terminal fragment of amyloid precursor protein downstream of the beta-secretase cleavage site. Cleavage of C100Flag by gamma-secretase was detected by electrochemiluminescence using antibodies that specifically recognize the Abeta40 or Abeta42 termini. Incubation of C100Flag with HeLa cell membranes or detergent-solubilized HeLa cell membranes generates both the Abeta40 and Abeta42 termini. Recovery of catalytically competent, soluble gamma-secretase critically depends on the choice of detergent; CHAPSO (3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate) but not Triton X-100 is suitable. Solubilized gamma-secretase activity is inhibited by pepstatin and more potently by a novel aspartyl protease transition-state analog inhibitor that blocks formation of Abeta40 and Abeta42 in mammalian cells. Upon gel exclusion chromatography, solubilized gamma-secretase activity coelutes with presenilin 1 (PS1) at an apparent relative molecular weight of approximately 2.0 x 10(6). Anti-PS1 antibody immunoprecipitates gamma-secretase activity from the solubilized gamma-secretase preparation. These data suggest that gamma-secretase activity is catalyzed by a PS1-containing macromolecular complex.
Li
Y M
YM
Department of Biological Chemistry, Merck Research Laboratories, West Point, PA 19486, USA. yueming_li@merck.com
Lai
M T
MT
Xu
M
M
Huang
Q
Q
DiMuzio-Mower
J
J
Sardana
M K
MK
Shi
X P
XP
Yin
K C
KC
Shafer
J A
JA
Gardell
S J
SJ
D000544:Alzheimer Disease / Q000201:enzymology; D053829:Amyloid Precursor Protein Secretases; D016564:Amyloid beta-Protein Precursor / Q000378:metabolism; D016282:Aspartic Acid Endopeptidases; D002219:Carbamates / Q000494:pharmacology; D002458:Cell Fractionation / Q000379:methods; D002462:Cell Membrane / Q000737:chemistry; D002793:Cholic Acids / Q000494:pharmacology; D003902:Detergents / Q000494:pharmacology; D004151:Dipeptides / Q000494:pharmacology; D010450:Endopeptidases / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D006367:HeLa Cells / Q000737:chemistry / Q000187:drug effects; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D009363:Neoplasm Proteins / Q000302:isolation & purification; D010436:Pepstatins / Q000494:pharmacology; D053764:Presenilin-1; D011480:Protease Inhibitors / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012995:Solubility; D013379:Substrate Specificity
D016564:Amyloid beta-Protein Precursor; D002219:Carbamates; D002793:Cholic Acids; D003902:Detergents; D004151:Dipeptides; C410009:L 685458; D008565:Membrane Proteins; D009363:Neoplasm Proteins; C508853:PSEN1 protein, human; D010436:Pepstatins; D053764:Presenilin-1; D011480:Protease Inhibitors; D011993:Recombinant Fusion Proteins; C014195:Streptomyces pepsin inhibitor; C048531:chapso; D053829:Amyloid Precursor Protein Secretases; D010450:Endopeptidases; D016282:Aspartic Acid Endopeptidases; C509280:BACE1 protein, human; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate; C031375:pepstatin