In Vitro Characterization of the Presenilin-Dependent γ-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.

Characterization of presenilin complexes from mouse and human brain using Blue Native gel electrophoresis reveals high expression in embryonic brain and minimal change in complex mobility with pathogenic presenilin mutations

“…Alternative approaches have provided evidence that PS and cofactors interact directly: affinity isolation of the complexes [9], coimmunoprecipitation studies [12,[37][38][39], and complex isolation with c-secretase inhibitor [4,22,54]. Other recent studies have also utilized BN/PAGE for analysis of PS complexes.…”

“…Synthetic peptides derived from human sequences were conjugated to diphtheria toxoid and rabbit antibodies generated as follows: Ab 98/1, PS1 NT (1-20) [20]; Ab 00/ 1 and Ab 00/2 PS1 loop (301-317) [21]; Ab 00/12, PS2 loop (307-336); Ab 00/19 NCT CT (691-709]) [22]; Ab 00/22 NCT ectodomain (331-346); Ab 02/45 PEN-2 NT (1-15) and Ab 02/41 APH-1b CT (244-257). Ab 00/6 was raised to human BACE CT (485-501) [23].…”

Pen2 and Presenilin-1 Modulate the Dynamic Equilibrium of Presenilin-1 and Presenilin-2 γ-Secretase Complexes

“…It is known that only a small percentage of PS is engaged in catalytically active complexes (35,36). Therefore, traditional Western blotting and immunoprecipitation methods would be inadequate for our study because they fail to distinguish between catalytically active and inactive complexes.…”

“…To overcome this, we attempted to develop small molecular affinity probes that would allow us to capture and characterize the active ␥-secretase complex under native conditions. L458 is a potent transition state analog that selectively binds to catalytically active ␥-secretase, and its analogs have been used to characterize ␥-secretase (17,36) (Fig. 3a).…”

C-terminal Fragment of Presenilin Is the Molecular Target of a Dipeptidic γ-Secretase-specific Inhibitor DAPT (N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-Butyl Ester)

“…Transition-state analogue-type aspartyl protease inhibitors bind directly to these PS fragments (6,7), suggesting that PS is the catalytic component of ␥-secretase. Intriguingly, immobilized transition-state analogue inhibitors, which are predicted to occupy the catalytic site, can copurify ␥-secretase with its ␤-amyloid precursor protein-derived natural substrate, C83 (8,9). Furthermore, these inhibitors unexpectedly displayed a noncompetitive pharmacological inhibition profile (10).…”