Purification and characterization of the human gamma-secretase complex.
Gamma-secretase is a member of an unusual class of proteases with intramembrane catalytic sites. This enzyme cleaves many type I membrane proteins, including the amyloid beta-protein (Abeta) precursor (APP) and the Notch receptor. Biochemical and genetic studies have identified four membrane proteins as components of gamma-secretase: heterodimeric presenilin (PS) composed of its N- and C-terminal fragments (PS-NTF/CTF), a mature glycosylated form of nicastrin (NCT), Aph-1, and Pen-2. Recent data from studies in Drosophila, mammalian, and yeast cells suggest that PS, NCT, Aph-1, and Pen-2 are necessary and sufficient to reconstitute gamma-secretase activity. However, many unresolved issues, in particular the possibility of other structural or regulatory components, would be resolved by actually purifying the enzyme. Here, we report a detailed, multistep purification procedure for active gamma-secretase and an initial characterization of the purified protease. Extensive mass spectrometry of the purified proteins strongly suggests that PS-NTF/CTF, mNCT, Aph-1, and Pen-2 are the components of active gamma-secretase. Using the purified gamma-secretase, we describe factors that modulate the production of specific Abeta species: (1) phosphatidylcholine and sphingomyelin dramatically improve activity without changing cleavage specificity within an APP substrate; (2) increasing CHAPSO concentrations from 0.1 to 0.25% yields a approximately 100% increase in Abeta42 production; (3) exposure of an APP-based recombinant substrate to 0.5% SDS modulates cleavage specificity from a disease-mimicking pattern (high Abeta42/43) to a physiological pattern (high Abeta40); and (4) sulindac sulfide directly and preferentially decreases Abeta42 cleavage within the purified complex. Taken together, our results define a procedure for purifying active gamma-secretase and suggest that the lipid-mediated conformation of both enzyme and substrate regulate the production of the potentially neurotoxic Abeta42 and Abeta43 peptides.
Center for Neurologic Diseases, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA.
Patrick C
PC
Fraering
Wenjuan
W
Ye
Jean-Marc
JM
Strub
Georgia
G
Dolios
Matthew J
MJ
LaVoie
Beth L
BL
Ostaszewski
Alain
A
van Dorsselaer
Rong
R
Wang
Dennis J
DJ
Selkoe
Michael S
MS
Wolfe
D000595:Amino Acid Sequence; D053829:Amyloid Precursor Protein Secretases; D016229:Amyloid beta-Peptides / Q000378:metabolism; D000818:Animals; D016282:Aspartic Acid Endopeptidases; D016466:CHO Cells; D006224:Cricetinae; D010450:Endopeptidases / Q000737:chemistry* / Q000302:isolation & purification*; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008055:Lipids / Q000737:chemistry; D046911:Macromolecular Substances; D013058:Mass Spectrometry; D008562:Membrane Glycoproteins / Q000302:isolation & purification; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D009842:Oligopeptides; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D010447:Peptide Hydrolases; D010455:Peptides / Q000737:chemistry; D053764:Presenilin-1; D011480:Protease Inhibitors / Q000737:chemistry; D051880:Receptors, Notch; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry; D013379:Substrate Specificity; D013467:Sulindac / Q000031:analogs & derivatives* / Q000737:chemistry; D014280:Triglycerides / Q000737:chemistry; D005680:gamma-Aminobutyric Acid / Q000031:analogs & derivatives* / Q000737:chemistry
D016229:Amyloid beta-Peptides; D008055:Lipids; D046911:Macromolecular Substances; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D009842:Oligopeptides; C508853:PSEN1 protein, human; C469349:PSENEN protein, human; D010446:Peptide Fragments; D010455:Peptides; D053764:Presenilin-1; D011480:Protease Inhibitors; D051880:Receptors, Notch; D014280:Triglycerides; C078592:amyloid beta-protein (1-40); C075222:amyloid beta-protein (1-42); C413604:nicastrin protein; D013467:Sulindac; D012967:Sodium Dodecyl Sulfate; D005680:gamma-Aminobutyric Acid; C025462:sulindac sulfide; C057077:1,2-dilinolenoyl-3-(4-aminobutyryl)propane-1,2,3-triol; C085706:FLAG peptide; C475154:APH1A protein, human; D053829:Amyloid Precursor Protein Secretases; D010450:Endopeptidases; D010447:Peptide Hydrolases; D016282:Aspartic Acid Endopeptidases; C509280:BACE1 protein, human; C509282:Bace1 protein, mouse