In vitro characterization of the presenilin-dependent gamma-secretase complex using a novel affinity ligand.
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.
Beher
Dirk
D
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
Fricker
Michael
M
Nadin
Alan
A
Clarke
Earl E
EE
Wrigley
Jonathan D J
JD
Li
Yue-Ming
YM
Culvenor
Janetta G
JG
Masters
Colin L
CL
Harrison
Timothy
T
Shearman
Mark S
MS
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
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