title: In vitro characterization of the presenilin-dependent gamma-secretase complex using a novel affinity ligand.
abstract: Gamma-secretase is the enzyme activity releasing the amyloid-beta peptide from membrane-bound processing intermediates derived from the beta-amyloid precursor protein. Cellular release and subsequent aggregation of the amyloid-beta peptide is thought to be causative for the pathogenesis of Alzheimer's disease. Gamma-secretase performs an unusual intramembranous cleavage and has been closely linked to a macromolecular complex containing presenilins. To generate a molecular probe for gamma-secretase, we have developed a novel biotinylated affinity ligand which is based on a specific inhibitor containing a hydroxyethylene dipeptide isostere, known to serve as a transition state analogue for aspartic proteinases. Using this probe we confirmed the presence of the presenilin heterodimer and mature nicastrin in the active enzyme complex and, furthermore, that substrate binding site(s) and active center(s) are spatially separated. Affinity precipitations suggest that only a discrete fraction of cellular presenilin is present in the active gamma-secretase complex and that both gamma(40)- and gamma(42)-activities are mediated by the same molecular entity. This was also reflected by a co-distribution of both enzyme activities in subcellular fractions enriched for trans-Golgi network membranes.
authors.affiliation: Department of Biochemistry & Molecular Biology, Merck Sharp & Dohme Research Laboratories, The Neuroscience Research Centre, Terlings Park, Harlow, Essex CM20 2QR, United Kingdom. dirk_beher@merck.com
authors.forename: Dirk
authors.initials: D
authors.lastname: Beher
authors.forename: Michael
authors.initials: M
authors.lastname: Fricker
authors.forename: Alan
authors.initials: A
authors.lastname: Nadin
authors.forename: Earl E
authors.initials: EE
authors.lastname: Clarke
authors.forename: Jonathan D J
authors.initials: JD
authors.lastname: Wrigley
authors.forename: Yue-Ming
authors.initials: YM
authors.lastname: Li
authors.forename: Janetta G
authors.initials: JG
authors.lastname: Culvenor
authors.forename: Colin L
authors.initials: CL
authors.lastname: Masters
authors.forename: Timothy
authors.initials: T
authors.lastname: Harrison
authors.forename: Mark S
authors.initials: MS
authors.lastname: Shearman
mesh_terms: D053829:Amyloid Precursor Protein Secretases; D016282:Aspartic Acid Endopeptidases; D015153:Blotting, Western; D010450:Endopeptidases / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D008024:Ligands; D008565:Membrane Proteins / Q000378:metabolism*; D053764:Presenilin-1; D053766:Presenilin-2; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured
chemical_list: D008024:Ligands; D008565:Membrane Proteins; C508853:PSEN1 protein, human; C508861:PSEN2 protein, human; D053764:Presenilin-1; D053766:Presenilin-2; D053829:Amyloid Precursor Protein Secretases; D010450:Endopeptidases; D016282:Aspartic Acid Endopeptidases; C509280:BACE1 protein, human