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.
[Show abstract] [Hide abstract]
ABSTRACT: Many transmembrane helices contain serine and/or threonine residues whose side chains form intrahelical H-bonds with upstream carbonyl oxygens. Here, we investigated the impact of threonine side-chain/main-chain backbonding on the backbone dynamics of the amyloid precursor protein transmembrane helix. This helix consists of a N-terminal dimerization region and a C-terminal cleavage region, which is processed by γ-secretase to a series of products. Threonine mutations within this transmembrane helix are known to alter the cleavage pattern, which can lead to early-onset Alzheimer's disease. Circular dichroism spectroscopy and amide exchange experiments of synthetic transmembrane domain peptides reveal that mutating threonine enhances the flexibility of this helix. Molecular dynamics simulations show that the mutations reduce intrahelical amide H-bonding and H-bond lifetimes. In addition, the removal of side-chain/main-chain backbonding distorts the helix, which alters bending and rotation at a diglycine hinge connecting the dimerization and cleavage regions. We propose that the backbone dynamics of the substrate profoundly affects the way by which the substrate is presented to the catalytic site within the enzyme. Changing this conformational flexibility may thus change the pattern of proteolytic processing.Biophysical Journal 03/2014; 106(6):1318-26. DOI:10.1016/j.bpj.2014.02.013 · 3.83 Impact Factor
[Show abstract] [Hide abstract]
ABSTRACT: Alzheimer’s disease (AD) is characterized pathologically by an abundance of extracellular neuritic plaques composed primarily of the 42-amino acid amyloid β peptide variant (Aβ42). In the majority of familial AD (FAD) cases, e.g., those harboring mutations in presenilin 1 (PS1), there is a relative increase in the levels of Aβ42 compared to the levels of Aβ40. We previously reported the characterization of a series of aminothiazole-bridged aromates termed aryl aminothiazole γ-secretase modulators or AGSMs [Kounnas, M. Z., et al. (2010) Neuron 67, 769–780] and showed their potential for use in the treatment of FAD [Wagner, S. L., et al. (2012) Arch. Neurol. 69, 1255–1258]. Here we describe a series of GSMs with physicochemical properties improved compared to those of AGSMs. Specific heterocycle replacements of the phenyl rings in AGSMs provided potent molecules with improved aqueous solubilities. A number of these soluble γ-secretase modulators (SGSMs) potently lowered Aβ42 levels without inhibiting proteolysis of Notch or causing accumulation of amyloid precursor protein carboxy-terminal fragments, even at concentrations approximately 1000-fold greater than their IC50 values for reducing Aβ42 levels. The effects of one potent SGSM on Aβ peptide production were verified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry, showing enhanced production of a number of carboxy-truncated Aβ species. This SGSM also inhibited Aβ42 peptide production in a highly purified reconstituted γ-secretase in vitro assay system and retained the ability to modulate γ-secretase-mediated proteolysis in a stably transfected cell culture model overexpressing a human PS1 mutation validating the potential for use in FAD.Biochemistry 01/2014; 53(4):702–713. DOI:10.1021/bi401537v · 3.19 Impact Factor
Journal of Alzheimer's disease: JAD 01/2013; 33(3):547-658. · 3.61 Impact Factor