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Li, Y. M. et al. Photoactivated -secretase inhibitors directed to the active site covalently label presenilin 1. Nature 405, 689-694

Department of Biological Chemistry, Merck Research Laboratories, West Point, Pennsylvania 19486, USA.
Nature (Impact Factor: 42.35). 07/2000; 405(6787):689-94. DOI: 10.1038/35015085
Source: PubMed

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.

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    • "Activity-Based Photoaffinity Labeling and Capture Activity-based photoaffinity labeling was performed with breast cancer cell lines in 12-well tissue culture dish with 10 nM of JC-8 (Li et al., 2000; Shelton et al., 2009b). Capture of the active g-secretase complex was first performed under nondenaturing conditions using Compound 3 (Placanica et al., 2009). "
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    ABSTRACT: γ-Secretase is composed of four proteins that are obligatory for protease activity: presenilin, nicastrin, Aph1, and Pen-2. Despite the progress toward understanding the function of these individual subunits, there is no information available pertaining to the modulation of γ-secretase in response to environmental changes in cells. Here, we show that hypoxia upregulates γ-secretase activity through a direct interaction with Hif-1α, revealing an unconventional function for Hif-1α as an enzyme subunit, which is distinct from its canonical role as a transcription factor. Moreover, hypoxia-induced cell invasion and metastasis are alleviated by either γ-secretase inhibitors or a dominant-negative Notch coactivator, indicating that γ-secretase/Notch signaling plays an essential role in controlling these cellular processes. The present study reveals a mechanism in which γ-secretase can achieve temporal control through conditional interactions with regulatory proteins, such as Hif-1α, under select physiological and pathological conditions.
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    • "L-685,458 and fenofibrate were purchased from Bachem and SIGMA, respectively. L-852,646 [37] was kindly provided from Dr. Y. Li (Sloan-Kettering Cancer Center). Synthetic longer Aβ peptides (i.e., β-amyloid (1-43, #23573), (1-45, #61956-01), (1-46, #62076-01), (1-48, #61965-01), (1-49, #61963-01) were purchased from Anaspec. "
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    ABSTRACT: Amyloid-beta peptide ending at 42nd residue (Abeta42) is believed as a pathogenic peptide for Alzheimer disease. Although gamma-secretase is a responsible protease to generate Abeta through a processive cleavage, the proteolytic mechanism of gamma-secretase at molecular level is poorly understood. We found that the transmembrane domain (TMD) 1 of presenilin (PS) 1, a catalytic subunit for the gamma-secretase, as a key modulatory domain for Abeta42 production. Abeta42-lowering and -raising gamma-secretase modulators (GSMs) directly targeted TMD1 of PS1 and affected its structure. A point mutation in TMD1 caused an aberrant secretion of longer Abeta species including Abeta45 that are the precursor of Abeta42. We further found that the helical surface of TMD1 is involved in the binding of Abeta45/48 and that the binding was altered by GSMs as well as TMD1 mutation. Binding between PS1 TMD1 and longer Abeta is critical for Abeta42 production.
    Molecular Neurodegeneration 01/2014; 9(1):7. DOI:10.1186/1750-1326-9-7 · 5.29 Impact Factor
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    • "Aspartyl protease transition state analogue inhibitors are useful tools for functional studies of γ-secretase. One such compound is L-685,458, which potently inhibits γ-secretase activity [14], [15] and signal peptide peptidase [16]. Our group previously designed an L-685,458-based compound for the efficient affinity purification of γ-secretase and its interacting proteins [17]. "
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    ABSTRACT: Here, we present a highly sensitive method to study protein-protein interactions and subcellular location selectively for active multicomponent enzymes. We apply the method on γ-secretase, the enzyme complex that catalyzes the cleavage of the amyloid precursor protein (APP) to generate amyloid β-peptide (Aβ), the major causative agent in Alzheimer disease (AD). The novel assay is based on proximity ligation, which can be used to study protein interactions in situ with very high sensitivity. In traditional proximity ligation assay (PLA), primary antibody recognition is typically accompanied by oligonucleotide-conjugated secondary antibodies as detection probes. Here, we first performed PLA experiments using antibodies against the γ-secretase components presenilin 1 (PS1), containing the catalytic site residues, and nicastrin, suggested to be involved in substrate recognition. To selectively study the interactions of active γ-secretase, we replaced one of the primary antibodies with a photoreactive γ-secretase inhibitor containing a PEG linker and a biotin group (GTB), and used oligonucleotide-conjugated streptavidin as a probe. Interestingly, significantly fewer interactions were detected with the latter, novel, assay, which is a reasonable finding considering that a substantial portion of PS1 is inactive. In addition, the PLA signals were located more peripherally when GTB was used instead of a PS1 antibody, suggesting that γ-secretase matures distal from the perinuclear ER region. This novel technique thus enables highly sensitive protein interaction studies, determines the subcellular location of the interactions, and differentiates between active and inactive γ-secretase in intact cells. We suggest that similar PLA assays using enzyme inhibitors could be useful also for other enzyme interaction studies.
    PLoS ONE 05/2013; 8(5):e63962. DOI:10.1371/journal.pone.0063962 · 3.23 Impact Factor
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