␥-Secretase is a multi-component enzyme complex that performs an intramembranous cleavage, releasing amyloid- (A) peptides from processing intermediates of the -amyloid precursor protein. Because A peptides are thought to be causative for Alzheimer's disease, inhibiting ␥-secretase represents a potential treatment for this neurodegenerative condition. Whereas inhibitors directed at the active center of ␥-secretase inhibit the cleavage of all its substrates, certain non-steroidal antiinflammatory drugs (NSAIDs) have been shown to selectively reduce the production of the more amyloidogenic A(1-42) peptide without inhibiting alternative cleavages. In contrast to the majority of previous studies, however, we demonstrate that in cell-free systems the mode of action of selected NSAIDs and their derivatives, depending on the concentrations used, can either be classified as modulatory or inhibitory. At modulatory concentrations, a selective and, with respect to the substrate, noncompetitive inhibition of A(1-42) production was observed. At inhibitory concentrations, on the other hand, biochemical readouts reminiscent of a nonselective ␥-secretase inhibition were obtained. When these compounds were analyzed for their ability to displace a radiolabeled, transition-state analog inhibitor from solubilized enzyme, noncompetitive antagonism was observed. The allosteric nature of radioligand displacement suggests that NSAID-like inhibitors change the conformation of the ␥-secretase enzyme complex by binding to a novel site, which is discrete from the binding site for transition-state analogs and therefore distinct from the catalytic center. Consequently, drug discovery efforts aimed at this site may identify novel allosteric inhibitors that could benefit from a wider window for inhibition of ␥ (42)-cleavage over alternative cleavages in the -amyloid precursor protein and, more importantly, alternative substrates.According to the "amyloid cascade hypothesis" an enhanced production or decreased clearance of toxic amyloid- (A) 1 peptides is thought to be the cause of Alzheimer's disease (AD) (1). A peptides are processing products (2) of the type I transmembrane protein -amyloid precursor protein (APP) (3), which has undergone sequential cleavages by -and ␥-secretase enzymes. A common denominator (reviewed by Hardy (4)) for mutations causative of familial AD (FAD) has been revealed, being abnormalities in the metabolism of APP that appear to lead to an elevation of the production of the A(1-42) peptide species. This C-terminally elongated A peptide is more prone to aggregation than the shorter and more abundant A(1-40) species. Consequently, the prevention of A production by inhibiting either of the proteases required for processing of APP is currently viewed as a promising approach toward a therapy for AD. The membrane-bound aspartyl protease -site APP-cleaving enzyme 1 (5, 6) is the major -secretase required for the generation of A peptides. -Site APP-cleaving enzyme 1 has been shown to cleave within th...
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.
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. 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 ␥-secretase has been established. Here we describe for the first time that certain potent ␥-secretase inhibitors are able to interfere with the endoproteolytic processing of presenilin 1 (PS1). In addition, we identified a novel ␥-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 tertbutyl 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/␥-secretase complex assembly or trafficking. Furthermore, in cellbased systems CBAP does not completely recapitulate PS1 loss-of-function phenotypes. Even though the -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.
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