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.
Fraering
Patrick C
PC
Center for Neurologic Diseases, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA.
Ye
Wenjuan
W
Strub
Jean-Marc
JM
Dolios
Georgia
G
LaVoie
Matthew J
MJ
Ostaszewski
Beth L
BL
van Dorsselaer
Alain
A
Wang
Rong
R
Selkoe
Dennis J
DJ
Wolfe
Michael S
MS
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