Abstract
Intramembranous cleavage of the beta-amyloid precursor protein by gamma-secretase is the final processing event generating amyloid-beta peptides, which are thought to be causative agents for Alzheimer's disease. Missense mutations in the presenilin genes co-segregate with early-onset Alzheimer's disease, and, recently, a close biochemical linkage between presenilins and the identity of gamma-secretase has been established. Here we describe for the first time that certain potent gamma-secretase inhibitors are able to interfere with the endoproteolytic processing of presenilin 1 (PS1). In addition, we identified a novel gamma-secretase inhibitor, [1S-benzyl-4R-[1-(5-cyclohexyl-2-oxo-2,3-dihydro-1H-benzo[e][1,4]diazepin-3(R,S)-ylcarbamoyl)-S-ethylcarbamoyl]-2R-hydroxy-5-phenyl-pentyl]-carbamic acid tert-butyl ester (CBAP), which not only physically interacts with PS1, but upon chronic treatment produces a "pharmacological knock-down" of PS1 fragments. This indicates that the observed accumulation of full-length PS1 is caused by a direct inhibition of its endoproteolysis. The subsequent use of CBAP as a biological tool to increase full-length PS1 levels in the absence of exogenous PS1 expression has provided evidence that wild-type PS1 endoproteolysis is not required either for PS1/gamma-secretase complex assembly or trafficking. Furthermore, in cell-based systems CBAP does not completely recapitulate PS1 loss-of-function phenotypes. Even though the beta-amyloid precursor protein cleavage and the S3 cleavage of the Notch receptor are inhibited by CBAP, an impairment of Trk receptor maturation was not observed.
Highlights
Intramembranous cleavage of the -amyloid precursor protein by ␥-secretase is the final processing event generating amyloid- peptides, which are thought to be causative agents for Alzheimer’s disease
Structure-Activity Relationship for Inhibition of presenilin 1 (PS1) Endoproteolysis and ␥-Secretase Activity—Following our initial observation that treatment of SH-SY5Y cells for 16 h with 10 M amount of the specific ␥-secretase inhibitor L-685,458 results in an increase in PS1-FL steady-state levels (Fig. 1A), we compared a variety of derivatives for their potential to inhibit PS1-FL endoproteolysis with their ability to inhibit APP ␥-secretase activity, monitored by inhibition of A peptide production (Fig. 1)
Compound 7 (CBAP), a chimeric molecule containing the L-685,458 isostere and a benzodiazepine group, was the only compound identified of a large number of inhibitors (n Ϸ 70; Fig. 1 and data not shown) that, under the standard experimental protocol, produced a strong increase in PS1-FL levels while concomitantly causing a detectable reduction in the levels of PS1-NTF (Fig. 1B)
Summary
Intramembranous cleavage of the -amyloid precursor protein by ␥-secretase is the final processing event generating amyloid- peptides, which are thought to be causative agents for Alzheimer’s disease. We identified a novel ␥-secretase inhibitor, {1S-benzyl-4R-[1-(5-cyclohexyl-2-oxo-2,3-dihydro-1Hbenzo[e][1,4]diazepin-3(R,S)-ylcarbamoyl)-S-ethylcarbamoyl]-2R-hydroxy-5-phenyl-pentyl}-carbamic acid tertbutyl ester (CBAP), which physically interacts with PS1, but upon chronic treatment produces a “pharmacological knock-down” of PS1 fragments This indicates that the observed accumulation of full-length PS1 is caused by a direct inhibition of its endoproteolysis. We provide evidence that the proteolytic activity associated with PS1/␥-secretase necessary for the cleavage of APP and the S3 cleavage of the Notch receptor can be dissociated from its function in membrane protein trafficking. This suggests that phenotypes resulting from the absence of presenilin expression are not completely recapitulated by ␥-secretase inhibition
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