Photoactivated gamma-secretase inhibitors directed to the active site covalently label presenilin 1.
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.
- SourceAvailable from: Cheng-jun Hu[Show abstract] [Hide abstract]
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.
Article: The Genetics of Alzheimer's Disease[Show abstract] [Hide abstract]
ABSTRACT: Alzheimer's disease is a progressive, neurodegenerative disease that represents a growing global health crisis. Two major forms of the disease exist: early onset (familial) and late onset (sporadic). Early onset Alzheimer's is rare, accounting for less than 5% of disease burden. It is inherited in Mendelian dominant fashion and is caused by mutations in three genes (APP, PSEN1, and PSEN2). Late onset Alzheimer's is common among individuals over 65 years of age. Heritability of this form of the disease is high (79%), but the etiology is driven by a combination of genetic and environmental factors. A large number of genes have been implicated in the development of late onset Alzheimer's. Examples that have been confirmed by multiple studies include ABCA7, APOE, BIN1, CD2AP, CD33, CLU, CR1, EPHA1, MS4A4A/MS4A4E/MS4A6E, PICALM, and SORL1. Despite tremendous progress over the past three decades, roughly half of the heritability for the late onset of the disease remains unidentified. Finding the remaining genetic factors that contribute to the development of late onset Alzheimer's disease holds the potential to provide novel targets for treatment and prevention, leading to the development of effective strategies to combat this devastating disease.12/2012; 2012:246210. DOI:10.6064/2012/246210
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ABSTRACT: Genetic deletion or mutations of presenilin genes (PS1/PS2) cause familial Alzheimer's disease and calcium (Ca²⁺) signaling abnormalities. PS1/PS2 act as endoplasmic reticulum (ER) Ca²⁺ leak channels that facilitate passive Ca²⁺ leak across ER membrane. Studies with PS1/PS2 double knockout (PS1/PS2-DKO) mouse embryonic fibroblasts showed that PS1/PS2 were responsible for 80% of passive Ca²⁺ leak from the lumen of endoplasmic reticulum to cytosol. Transient transfection of the wild type PS1 expression construct increased cytoplasmic Ca²⁺ as a result of Ca²⁺ leak across ER membrane whereas the FADPS1 (PS1-M146V) mutation construct alone or in combination with the wild type PS1 expression construct abrogated Ca²⁺ leak in SK-N-SH cells. Inhibition of basal c-jun-NH2-terminal kinase (JNK) activity by JNK inhibitor SP600125 repressed PS1 transcription and PS1 protein expression by augmenting p53 protein level in SK-N-SH cells (Lee and Das 2008). In this report we also showed that repression of PS1 transcription by JNK inhibitor SP600125 inhibited passive Ca²⁺ leak across ER membrane which could be rescued by expressing PS1 wild type and not by expressing FADPS1 (PS1-M146V) under a SP600125 non-responsive promoter. Treatment of SK-N-SH cells with SP600125 also triggered InsP3R-mediated Ca²⁺ release from the ER by addition of 500 nM bradykinin, an agonist of InsP3 receptor (InsP3R1) without changing the expression of InsP3R1. This data confirms that SP600125 increases the Ca²⁺ store in the ER by inhibiting PS1-mediated Ca²⁺ leak across ER membrane. p53, ZNF237 and Chromodomain helicase DNA-binding protein 3 which are repressors of PS1 transcription, also reduced Ca²⁺ leak across ER membrane in SK-N-SH cells but γ-secretase inhibitor or dominant negative γ-secretase-specific PS1 mutant (PS1-D257A) had no significant effect. Therefore, p53, ZNF237, and Chromodomain helicase DNA-binding protein 3 inhibit the function ER Ca²⁺ leak channels to regulate both ER and cytoplasmic Ca²⁺ levels and may potentially control Ca²⁺-signaling function of PS1.Journal of Neurochemistry 05/2012; 122(3):487-500. DOI:10.1111/j.1471-4159.2012.07794.x · 4.24 Impact Factor