Photoactivated γ-secretase inhibitors directed to the active site covalently label presenilin 1

Abstract

Cleavage of amyloid precursor protein (APP) by the beta- and gamma-secretases generates the amino and carboxy termini, respectively, of the A beta amyloidogenic peptides A beta40 and A beta42--the major constituents of the amyloid plaques in the brain parenchyma of Alzheimer's disease patients. There is evidence that the polytopic membrane-spanning proteins, presenilin 1 and 2 (PS1 and PS2), are important determinants of gamma-secretase activity: mutations in PS1 and PS2 that are associated with early-onset familial Alzheimer's disease increase the production of A beta42 (refs 4-6), the more amyloidogenic peptide; gamma-secretase activity is reduced in neuronal cultures derived from PS1-deficient mouse embryos; and directed mutagenesis of two conserved aspartates in transmembrane segments of PS1 inactivates the ability of gamma-secretase to catalyse processing of APP within its transmembrane domain. It is unknown, however, whether PS1 (which has little or no homology to any known aspartyl protease) is itself a transmembrane aspartyl protease or a gamma-secretase cofactor, or helps to colocalize gamma-secretase and APP. Here we report photoaffinity labelling of PS1 (and PS2) by potent gamma-secretase inhibitors that were designed to function as transition state analogue inhibitors directed to the active site of an aspartyl protease. This observation indicates that PS1 (and PS2) may contain the active site of gamma-secretase. Interestingly, the intact, single-chain form of wild-type PS1 is not labelled by an active-site-directed photoaffinity probe, suggesting that intact wild-type PS1 may be an aspartyl protease zymogen.

Presenilin mutations linked to familial Alzheimer's disease cause an imbalance in phosphatidylinositol 4,5-bisphosphate metabolism

“…1C). By comparison, A␤ production has been shown to be efficiently inhibited by treatment with these inhibitors at concentrations far below those used in our experiments (32,33). These data indicate that presenilin-associated proteolytic activity is dispensable for altered MIC channel activity in FAD mutant PS1-expressing cells.…”

Signal Peptide Peptidase and γ-Secretase Share Equivalent Inhibitor Binding Pharmacology

“…Compounds and Radioligands-L-685,458 (13), (Z-LL) 2 -ketone (38), and DAPT (39) Cell Culture-THP-1 cells were grown in roller bottles in RPMI 1640 medium containing L-glutamine (Invitrogen) and 10 M ␤-mercaptoethanol to a density of 1 ϫ 10 6 /ml. Cells were harvested by centrifugation, and cell pellets were quick-frozen in dry ice/ethanol and stored at Ϫ80°C prior to use.…”

“…The presenilin subunit appears to carry the active site of the enzyme because it contains aspartyl protease active-site motifs essential for ␥-secretase activity (10,11) and can be photolabeled using transition state and substrate analogs (12)(13)(14). In addition to the transition state analog inhibitors, there are also non-transition state ␥-secretase inhibitors that target presenilin (15,16).…”

Progressive neuronal loss and behavioral impairments of transgenic C57BL/6 inbred mice expressing the carboxy terminus of amyloid precursor protein

“…PS1 has been considered to be a therapeutic target for the treatment and the delayed expression of AD symptoms (Li et al, 2000;Esler and Wolfe, 2001). The PS1-deficient mice generated by the conventional knockout strategy were embryonic lethal (Wong et al, 1997).…”