title: Transition-state analogue gamma-secretase inhibitors stabilize a 900 kDa presenilin/nicastrin complex.
abstract: Gamma-secretase mediates the final step, which generates Alzheimer's disease Abeta amyloid protein, by cleaving the transmembrane domain of the amyloid-beta protein precursor. Four gene products, presenilin, nicastrin, APH-1, and PEN-2, are required for gamma-secretase activity that is contained within a high molecular mass complex. To further characterize gamma-secretase, we probed membranes from human neuroblastoma SH-SY5Y cells with gamma-secretase inhibitor biotin derivatives of L-685,458, pepstatin A, and the difluoro alcohol 1-Bt. These inhibitor derivatives bound and precipitated PS1 fragments from membrane CHAPSO extracts. Analysis of PS1 complexes by blue native gel electrophoresis and western blotting indicated that the CHAPSO extracts contained complexes of approximately 900, 500, and 400 kDa. With this technique, derivatives of the three inhibitors were detected only in association with the 900 kDa species. Size-exclusion chromatography showed that 13% of PS1 immunoreactivity extracted with CHAPSO was comprised within a >or=900 kDa species with the remaining eluting in fractions of 669-250 kDa but that most enzymatic activity was associated with the 900 kDa fractions. After treatment with L-685,458 inhibitor, 49% PS1 immunoreactivity was eluted in the 900 kDa fraction, supporting evidence that the inhibitor stabilized this complex. Subcellular fractionation of SH-SY5Y cells indicated that the 900 kDa complex was formed as PS1 and NCT matured through the secretory pathway and that enzymatic activity correlated with complex maturation. From these observations, we propose a model for the structure of active gamma-secretase that would consist of dimerization of 400-500 kDa subunits and be consistent with the apparent molecular mass of the complex.
authors.affiliation: Department of Pathology, The University of Melbourne, Parkville 3010, and The Mental Health Research Institute, Parkville 3052, Australia. gmevin@unimelb.edu.au
authors.forename: Geneviève
authors.initials: G
authors.lastname: Evin
authors.forename: Louise D
authors.initials: LD
authors.lastname: Canterford
authors.forename: David E
authors.initials: DE
authors.lastname: Hoke
authors.forename: Robyn A
authors.initials: RA
authors.lastname: Sharples
authors.forename: Janetta G
authors.initials: JG
authors.lastname: Culvenor
authors.forename: Colin L
authors.initials: CL
authors.lastname: Masters
mesh_terms: D053829:Amyloid Precursor Protein Secretases; D016282:Aspartic Acid Endopeptidases / Q000378:metabolism*; D001665:Binding Sites; D002219:Carbamates / Q000737:chemistry / Q000378:metabolism; D045744:Cell Line, Tumor; D002850:Chromatography, Gel; D003432:Cross-Linking Reagents / Q000378:metabolism; D003902:Detergents / Q000737:chemistry; D004151:Dipeptides / Q000737:chemistry / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism*; D004795:Enzyme Stability; D006801:Humans; D008562:Membrane Glycoproteins / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D008970:Molecular Weight; D046912:Multiprotein Complexes / Q000737:chemistry / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism; D010777:Photochemistry; D053764:Presenilin-1; D011480:Protease Inhibitors / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D012995:Solubility; D013347:Subcellular Fractions / Q000201:enzymology / Q000378:metabolism
chemical_list: D002219:Carbamates; D003432:Cross-Linking Reagents; D003902:Detergents; D004151:Dipeptides; C410009:L 685458; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D046912:Multiprotein Complexes; C508853:PSEN1 protein, human; D010446:Peptide Fragments; D053764:Presenilin-1; D011480:Protease Inhibitors; C413604:nicastrin protein; D053829:Amyloid Precursor Protein Secretases; D010450:Endopeptidases; D016282:Aspartic Acid Endopeptidases; C509280:BACE1 protein, human