Beta-amyloid precursor protein mutants respond to gamma-secretase modulators.
ABSTRACT Pathogenic generation of the 42-amino acid variant of the amyloid beta-peptide (Abeta) by beta- and gamma-secretase cleavage of the beta-amyloid precursor protein (APP) is believed to be causative for Alzheimer disease (AD). Lowering of Abeta(42) production by gamma-secretase modulators (GSMs) is a hopeful approach toward AD treatment. The mechanism of GSM action is not fully understood. Moreover, whether GSMs target the Abeta domain is controversial. To further our understanding of the mode of action of GSMs and the cleavage mechanism of gamma-secretase, we analyzed mutations located at different positions of the APP transmembrane domain around or within the Abeta domain regarding their response to GSMs. We found that Abeta(42)-increasing familial AD mutations of the gamma-secretase cleavage site domain responded robustly to Abeta(42)-lowering GSMs, especially to the potent compound GSM-1, irrespective of the amount of Abeta(42) produced. We thus expect that familial AD patients carrying mutations at the gamma-secretase cleavage sites of APP should respond to GSM-based therapeutic approaches. Systematic phenylalanine-scanning mutagenesis of this region revealed a high permissiveness to GSM-1 and demonstrated a complex mechanism of GSM action as other Abeta species (Abeta(41), Abeta(39)) could also be lowered besides Abeta(42). Moreover, certain mutations simultaneously increased Abeta(42) and the shorter peptide Abeta(38), arguing that the proposed precursor-product relationship of these Abeta species is not general. Finally, mutations of residues in the proposed GSM-binding site implicated in Abeta(42) generation (Gly-29, Gly-33) and potentially in GSM-binding (Lys-28) were also responsive to GSMs, a finding that may question APP substrate targeting of GSMs.
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ABSTRACT: The Amyloid Precursor Protein (APP) is widely expressed type-I transmembrane (TM) glycoprotein present at the neuronal synapse. The proteolytic cleavage by γ-secretase of its C-terminal fragment produces amyloid-β (Aβ) peptides of different lengths, the deposi- tion of which is an early indicator of Alzheimer's disease (AD). At present, there is no consensus on the conformation of the APP-TM domain at the biological membrane. Although structures have been determined by nuclear magnetic resonance (NMR) in deter- gent micelles, their conformation is markedly different. Here we show by using molecular simulations that the APP-TM region systematically prefers a straight α-helical conformation once embedded in a membrane bi- layer. APP-TM is highly flexible however, and its secondary structure is strongly influenced by the surrounding lipid environment, as when enclosed in detergent micelles. This behavior is confirmed when analyzing in silico the atomistic APP-TM population observed by residual dipolar couplings and double electron-electron resonance (DEER) spectroscopy. These structural and dynamic features are critical in the proteolytic pro- cessing of APP by the γ-secretase enzyme, as suggested by a series of G700 mutants. Affecting the hydration and flexibility of APP-TM these mutants show a correlated increase in the production of Aβ38 compared to Aβ40 peptides, which is reminiscent of the effect of γ-secretase modulators inhibitors.Journal of Biological Chemistry 01/2014; · 4.65 Impact Factor
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ABSTRACT: γ-Secretase generates the peptides of Alzheimer's disease, Aβ(40) and Aβ(42), by cleaving the amyloid precursor protein within its transmembrane domain. γ-Secretase also cleaves numerous other substrates, raising concerns about γ-secretase inhibitor off-target effects. Another important class of drugs, γ-secretase modulators, alter the cleavage site of γ-secretase on amyloid precursor protein, changing the Aβ(42)/Aβ(40) ratio, and are thus a promising therapeutic approach for Alzheimer's disease. However, the target for γ-secretase modulators is uncertain, with some data suggesting that they function on γ-secretase, whereas others support their binding to the amyloid precursor. In this paper we address this controversy by using a fluorescence resonance energy transfer-based assay to examine whether γ-secretase modulators alter Presenilin-1/γ-secretase conformation in intact cells in the absence of its natural substrates such as amyloid precursor protein and Notch. We report that the γ-secretase allosteric site is located within the γ-secretase complex, but substrate docking is needed for γ-secretase modulators to access this site.Nature Communications 11/2010; 1:130. · 10.74 Impact Factor
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ABSTRACT: The amyloid β precursor protein (APP) is a single-pass transmembrane glycoprotein that is ubiquitously expressed in many cell types, including neurons. Amyloidogenic processing of APP by β- and γ-secretases leads to the production of amyloid-β (Aβ) peptides that can oligomerize and aggregate into amyloid plaques, a characteristic hallmark of Alzheimer's disease (AD) brains. Multiple reports suggest that dimerization of APP may play a role in Aβ production; however, it is not yet clear whether APP dimers increase or decrease Aβ and the mechanism is not fully understood. To better understand the relationship between APP dimerization and production of Aβ, a high throughput screen for small molecule modulators of APP dimerization was conducted using APP-Firefly luciferase enzyme complementation to detect APP dimerization. Selected modulators identified from a compound library of 77,440 compounds were tested for their effects on Aβ generation. Two molecules that inhibited APP dimerization produced a reduction in Aβ levels as measured by ELISA. The inhibitors did not change sAPPα or γ-CTF levels, but lowered sAPPβ levels, suggesting that blocking the dimerization is preventing the cleavage by β-secretase in the amyloidogenic processing of APP. To our knowledge, this is the first High Throughput Screen (HTS) effort to identify small molecule modulators of APP dimerization. Inhibition of APP dimerization has previously been suggested as a therapeutic target in AD. The findings reported here further support that modulation of APP dimerization may be a viable means of reducing the production of Aβ.American journal of neurodegenerative disease. 05/2012; 1(1):75-87.