amyloid-beta-peptides and 1-2-dilinolenoyl-3-(4-aminobutyryl)propane-1-2-3-triol

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.

Topics: Amino Acid Sequence; Amyloid beta-Peptides; Amyloid Precursor Protein Secretases; Animals; Aspartic Acid Endopeptidases; CHO Cells; Cricetinae; Endopeptidases; gamma-Aminobutyric Acid; Humans; Hydrolysis; Kinetics; Lipids; Macromolecular Substances; Mass Spectrometry; Membrane Glycoproteins; Membrane Proteins; Mice; Molecular Sequence Data; Oligopeptides; Peptide Fragments; Peptide Hydrolases; Peptides; Presenilin-1; Protease Inhibitors; Receptors, Notch; Sodium Dodecyl Sulfate; Substrate Specificity; Sulindac; Triglycerides

The presenilin (PS) complex, including PS, nicastrin, APH-1 and PEN-2, is essential for gamma-secretase activity, which is required for amyloid beta-protein (Abeta) generation. However, the precise individual roles of the three cofactors in the PS complex in Abeta generation remain to be clarified. Here, to distinguish the roles of PS cofactors in gamma-secretase activity from those in PS endoproteolysis, we investigated their roles in the gamma-secretase activity reconstituted by the coexpression of PS N- and C-terminal fragments (NTF and CTF) in PS-null cells. We demonstrate that the coexpression of PS1 NTF and CTF forms the heterodimer and restores Abeta generation in PS-null cells. The generation of Abeta was saturable at a certain expression level of PS1 NTF/CTF, while the overexpression of PEN-2 alone resulted in a further increase in Abeta generation. Although PEN-2 did not enhance PS1 NTF/CTF heterodimer formation, PEN-2 expression reduced the IC50 of a specific gamma-secretase inhibitor, a transition state analogue, for Abeta generation, suggesting that PEN-2 expression enhances the affinity or the accessibility of the substrate to the catalytic site. Thus, our results strongly suggest that PEN-2 is not only an essential component of the gamma-secretase complex but also an enhancer of gamma-cleavage after PS heterodimer formation.

Topics: Alanine; Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Amyloid Precursor Protein Secretases; Animals; Aspartic Acid; Aspartic Acid Endopeptidases; Blotting, Western; Carbamates; Cell Line; Dipeptides; Dose-Response Relationship, Drug; Endopeptidases; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; Fibroblasts; gamma-Aminobutyric Acid; Gene Expression Regulation; Membrane Glycoproteins; Membrane Proteins; Mice; Peptide Fragments; Presenilin-1; Presenilin-2; Protein Processing, Post-Translational; Proto-Oncogene Proteins c-myc; Radioimmunoassay; Statistics, Nonparametric; Transgenes; Triglycerides

gamma-Secretase activity is involved in the generation of Abeta and therefore likely contributes to the pathology of Alzheimer's disease. Blocking this activity was seen as a major therapeutic target to slow down or arrest Abeta-related AD progression. This strategy seemed more doubtful when it was established that gamma-secretase also targets other substrates including Notch, a particularly important transmembrane protein involved in vital functions, at both embryonic and adulthood stages. We have described previously new non-peptidic inhibitors able to selectively inhibit Abeta cellular production in vitro without altering Notch pathway. We show here that in vivo, these inhibitors do not alter the Notch pathway responsible for somitogenesis in the zebrafish embryo. In addition, we document further the selectivity of JLK inhibitors by showing that, unlike other described gamma-secretase inhibitors, these agents do not affect E-cadherin processing. Finally, we establish that JLKs do not inhibit beta-site APP cleaving enzymes (BACE) 1 and BACE2, alpha-secretase, the proteasome, and GSK3beta kinase. Altogether, JLK inhibitors are the sole agents to date that are able to prevent Abeta production without triggering unwanted cleavages of other proteins.

Topics: Amyloid beta-Peptides; Amyloid Precursor Protein Secretases; Animals; Anticoagulants; Aspartic Acid Endopeptidases; Blotting, Western; Cadherins; Carbamates; Cell Line; Cysteine Endopeptidases; Dipeptides; Dose-Response Relationship, Drug; Embryo, Mammalian; Embryo, Nonmammalian; Endopeptidases; gamma-Aminobutyric Acid; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; In Situ Hybridization; In Vitro Techniques; Kidney; Membrane Proteins; Multienzyme Complexes; Mutation; Peptide Fragments; Precipitin Tests; Proteasome Endopeptidase Complex; Receptors, Notch; Time Factors; Transfection; Triglycerides; Zebrafish