Activity-Dependent Isolation of the Presenilin- γ-Secretase Complex Reveals Nicastrin and a γ Substrate

• Published in: Proceedings of the National Academy of Sciences - PNAS 2002-03, Vol.99 (5), p.2720-2725
• Main Authors: Esler, William P., Kimberly, W. Taylor, Ostaszewski, Beth L., Ye, Wenjuan, Diehl, Thekla S., Selkoe, Dennis J., Wolfe, Michael S.
• Format: Article
• Language: English
• Subjects: Active sites; Alzheimers disease; Amyloid beta-Protein Precursor - metabolism; Amyloid Precursor Protein Secretases; Animals; Antibodies; Aspartic Acid Endopeptidases - metabolism; Biochemistry; Biological Sciences; Chromatography; Detergents; Dimerization; Elution; Endopeptidases - metabolism; HeLa cells; Membrane Glycoproteins - metabolism; Membrane Proteins - metabolism; Mice; Microsomes; Models, Chemical; nicastrin; Presenilin-1; Presenilin-2; Proteins; Resins; Substrate Specificity
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.052436599

Presenilin heterodimers apparently contain the active site of γ-secretase, a polytopic aspartyl protease involved in the transmembrane processing of both the Notch receptor and the amyloid-β precursor protein. Although critical to embryonic development and the pathogenesis of Alzheimer's disease, this protease is difficult to characterize, primarily because it is a multicomponent complex of integral membrane proteins. Here the functional γ-secretase complex was isolated by using an immobilized active site-directed inhibitor of the protease. Presenilin heterodimers and nicastrin bound specifically to this inhibitor under conditions tightly correlating with protease activity, whereas several other presenilin-interacting proteins (β-catenin, calsenilin, and presenilin-associated protein) did not bind. Moreover, anti-nicastrin antibodies immunoprecipitated γ-secretase activity from detergent-solubilized microsomes. Unexpectedly, C83, the major endogenous amyloid-β precursor protein substrate of γ-secretase, was also quantitatively associated with the complex. These results provide direct biochemical evidence that nicastrin is a member of the active γ-secretase complex, indicate that β-catenin, calsenilin, and presenilin-associated protein are not required for γ activity, and suggest an unprecedented mechanism of substrate-protease interaction.