Phosphoinositides suppress gamma-secretase in both the detergent-soluble and -insoluble states.
ABSTRACT gamma-Secretase is an aspartic protease that hydrolyzes type I membrane proteins within the hydrophobic environment of the lipid bilayer. Using the CHAPSO-solubilized gamma-secretase assay system, we previously found that gamma-secretase activity was sensitive to the concentrations of detergent and phosphatidylcholine. This strongly suggests that the composition of the lipid bilayer has a significant impact on the activity of gamma-secretase. Recently, level of secreted beta-amyloid protein was reported to be attenuated by increasing levels of phosphatidylinositol 4,5-diphosphate (PI(4,5)P2) in cultured cells. However, it is not clear whether PI(4,5)P2 has a direct effect on gamma-secretase activity. In this study, we found that phosphoinositides directly inhibited CHAPSO-solubilized gamma-secretase activity. Interestingly, neither phosphatidylinositol nor inositol triphosphate altered gamma-secretase activity. PI(4,5)P2 was also found to inhibit gamma-secretase activity in CHAPSO-insoluble membrane microdomains (rafts). Kinetic analysis of beta-amyloid protein production in the presence of PI(4,5)P2 suggested a competitive inhibition. Even though phosphoinositides are minor phospholipids of the membrane, the concentration of PI(4,5)P2 within the intact membrane has been reported to be in the range of 4-8 mm. The presence of PI(4,5)P2-rich rafts in the membrane has been reported in a range of cell types. Furthermore, gamma-secretase is enriched in rafts. Taking these data together, we propose that phosphoinositides potentially regulate gamma-secretase activity by suppressing its association with the substrate.
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ABSTRACT: Sphingosine-1-phosphate (S1P) is a pluripotent lipophilic mediator working as a ligand for G-protein coupled S1P receptors (S1PR), which is currently highlighted as a therapeutic target for autoimmune diseases including relapsing forms of multiple sclerosis. Sphingosine related compounds, FTY720 and KRP203 known as S1PR modulators, are phosphorylated by sphingosine kinase 2 (SphK2) to yield the active metabolites FTY720-P and KRP203-P, which work as functional antagonists for S1PRs. Here we report that FTY720 and KRP203 decreased production of Amyloid-β peptide (Aβ), a pathogenic proteins causative for Alzheimer disease (AD), in cultured neuronal cells. Pharmacological analyses suggested that the mechanism of FTY720-mediated Aβ decrease in cells was independent of known downstream signaling pathways of S1PRs. Unexpectedly, 6-days treatment of APP transgenic mice with FTY720 resulted in a decrease in Aβ40, but an increase in Aβ42 levels in brains. These results suggest that S1PR modulators are novel type of regulators for Aβ metabolisms that are active in vitro and in vivo.PLoS ONE 01/2013; 8(5):e64050. · 3.73 Impact Factor
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ABSTRACT: Understanding the substrate recognition mechanism of γ-secretase is a key step for establishing substrate-specific inhibition of amyloid β-protein (Aβ) production. However, it is widely believed that γ-secretase is a promiscuous protease and that its substrate-specific inhibition is elusive. Here we show that γ-secretase distinguishes the ectodomain length of substrates and preferentially captures and cleaves substrates containing a short ectodomain. We also show that a subset of peptides containing the CDCYCxxxxCxCxSC motif binds to the amino terminus of C99 and inhibits Aβ production in a substrate-specific manner. Interestingly, these peptides suppress β-secretase-dependent cleavage of APP, but not that of sialyltransferase 1. Most importantly, intraperitoneal administration of peptides into mice results in a significant reduction in cerebral Aβ levels. This report provides direct evidence of the substrate preference of γ-secretase and its mechanism. Our results demonstrate that the ectodomain of C99 is a potent target for substrate-specific anti-Aβ therapeutics to combat Alzheimer's disease.Nature Communications 10/2013; 4:2529. · 10.02 Impact Factor
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ABSTRACT: Alzheimer's disease (AD) is neuropathologically characterized by the combined occurrence of extracellular beta-amyloid plaques and intracellular neurofibrillary tangles in the brain. While plaques contain aggregated forms of the amyloid beta-peptide (Abeta), tangles are formed by fibrillar forms of the microtubule associated protein tau. All mutations identified so far to cause familial forms of early onset AD (FAD) are localized close to or within the Abeta domain of the amyloid precursor protein (APP) or in the presenilin proteins that are essential components of a protease complex involved in the generation of Abeta. Mutations in the tau gene are not associated with FAD, but can cause other forms of dementia. The genetics of FAD together with biochemical and cell biological data, led to the formulation of the amyloid hypothesis, stating that accumulation and aggregation of Abeta is the primary event in the pathogenesis of AD, while tau might mediate its toxicity and neurodegeneration.The generation of Abeta involves sequential proteolytic cleavages of the amyloid precursor protein (APP) by enzymes called beta-and gamma-secretases. Notably, APP itself as well as the secretases are integral membrane proteins. Thus, it is very likely that membrane lipids are involved in the regulation of subcellular transport, activity, and metabolism of AD related proteins.Indeed, several studies indicate that membrane lipids, including cholesterol and sphingolipids (SLs) affect Abeta generation and aggregation. Interestingly, APP and other AD associated proteins, including beta-and gamma-secretases can, in turn, influence lipid metabolic pathways. Here, we review the close connection of cellular lipid metabolism and AD associated proteins and discuss potential mechanisms that could contribute to initiation and progression of AD.Molecular Neurodegeneration 10/2013; 8(1):34. · 4.01 Impact Factor