Satoko Osawa

Equimolar Production of Amyloid β-Protein and Amyloid Precursor Protein Intracellular Domain from β-Carboxyl-terminal Fragment by γ-Secretase

We showed previously that cells expressing wild-type (WT) β-amyloid precursor protein (APP) or coexpressing WTAPP and WT presenilin (PS) 1/2 produced APP intracellular domains (AICD) 49-99 and 50-99, with the latter predominating. On the other hand, the cells expressing mutant (MT) APP or coexpressing WTAPP and MTPS1/2 produced a greater proportion of AICD-(49-99) than AICD-(50-99). In addition, the expression of amyloid β-protein (Aβ) 49 in cells resulted in predominant production of Aβ40 and that of Aβ48 leads to preferential production of Aβ42. These observations suggest that ϵ-cleavage and γ-cleavage are interrelated. To determine the stoichiometry between Aβ and AICD, we have established a 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonic acid-solubilized γ-secretase assay system that exhibits high specific activity. By using this assay system, we have shown that equal amounts of Aβ and AICD are produced from β-carboxyl-terminal fragment (C99) by γ-secretase, irrespective of WT or MTAPP and PS1/2. Although various Aβ species, including Aβ40, Aβ42, Aβ43, Aβ45, Aβ48, and Aβ49, are generated, only two species of AICD, AICD-(49-99) and AICD-(50-99), are detected. We also have found that M233T MTPS1 produced only one species of AICD, AICD-(49-99), and only one for its counterpart, Aβ48, in contrast to WT and other MTPS1s. These strongly suggest thatϵ-cleavage is the primary event, and the produced Aβ48 and Aβ49 rapidly undergo γ-cleavage, resulting in generation of various Aβ species.

BACE1 Activity Is Modulated by Cell-Associated Sphingosine-1-Phosphate

Sphingosine kinase (SphK) 1 and 2 phosphorylate sphingosine to generate sphingosine-1-phosphate (S1P), a pluripotent lipophilic mediator implicated in a variety of cellular events. Here we show that the activity of β-site APP cleaving enzyme-1 (BACE1), the rate-limiting enzyme for amyloid-β peptide (Aβ) production, is modulated by S1P in mouse neurons. Treatment by SphK inhibitor, RNA interference knockdown of SphK, or overexpression of S1P degrading enzymes decreased BACE1 activity, which reduced Aβ production. S1P specifically bound to full-length BACE1 and increased its proteolytic activity, suggesting that cellular S1P directly modulates BACE1 activity. Notably, the relative activity of SphK2 was upregulated in the brains of patients with Alzheimers disease. The unique modulatory effect of cellular S1P on BACE1 activity is a novel potential therapeutic target for Alzheimers disease.

Phenylpiperidine‐type γ‐secretase modulators target the transmembrane domain 1 of presenilin 1

Amyloid‐β peptide ending at the 42nd residue (Aβ42) is implicated in the pathogenesis of Alzheimers disease (AD). Small compounds that exhibit selective lowering effects on Aβ42 production are termed γ‐secretase modulators (GSMs) and are deemed as promising therapeutic agents against AD, although the molecular target as well as the mechanism of action remains controversial. Here, we show that a phenylpiperidine‐type compound GSM‐1 directly targets the transmembrane domain (TMD) 1 of presenilin 1 (PS1) by photoaffinity labelling experiments combined with limited digestion. Binding of GSM‐1 affected the structure of the initial substrate binding and the catalytic sites of the γ‐secretase, thereby decreasing production of Aβ42, possibly by enhancing its conversion to Aβ38. These data indicate an allosteric action of GSM‐1 by directly binding to the TMD1 of PS1, pinpointing the target structure of the phenylpiperidine‐type GSMs.

Neutralization of the γ-secretase activity by monoclonal antibody against extracellular domain of nicastrin.

Several lines of evidence suggest that aberrant Notch signaling contributes to the development of several types of cancer. Activation of Notch receptor is executed through intramembrane proteolysis by γ-secretase, which is a multimeric membrane-embedded protease comprised of presenilin, nicastrin (NCT), anterior pharynx defective 1 and PEN-2. In this study, we report the neutralization of the γ-secretase activity by a novel monoclonal antibody A5226A against the extracellular domain of NCT, generated by using a recombinant budded baculovirus as an immunogen. This antibody recognized fully glycosylated mature NCT in the active γ-secretase complex on the cell surface, and inhibited the γ-secretase activity by competing with the substrate binding in vitro. Moreover, A5226A abolished the γ-secretase activity-dependent growth of cancer cells in a xenograft model. Our data provide compelling evidence that NCT is a molecular target for the mechanism-based inhibition of γ-secretase, and that targeting NCT might be a novel therapeutic strategy against cancer caused by aberrant γ-secretase activity and Notch signaling.

FTY720/Fingolimod, a Sphingosine Analogue, Reduces Amyloid-β Production in Neurons

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.

Allosteric regulation of γ-secretase activity by a phenylimidazole-type γ-secretase modulator

Significance For mechanism-based development of treatment for Alzheimer’s disease (AD), the precise molecular mechanism of γ-secretase modulators (GSMs), which have been extensively developed as possible therapeutic reagents, is required. Here, we analyzed the mode of actions of phenylimidazole-type GSMs using a chemical biology approach and systematic mutagenesis. We provide the first structural model, to our knowledge, that binding of the phenylimidazole-type GSMs at the luminal loop of presenilin induces a conformational change of the catalytic center to reduce toxic amyloid-β species selectively. Our results may facilitate the effective development of AD therapeutics. γ-Secretase is an intramembrane-cleaving protease responsible for the generation of amyloid-β (Aβ) peptides. Recently, a series of compounds called γ-secretase modulators (GSMs) has been shown to decrease the levels of long toxic Aβ species (i.e., Aβ42), with a concomitant elevation of the production of shorter Aβ species. In this study, we show that a phenylimidazole-type GSM allosterically induces conformational changes in the catalytic site of γ-secretase to augment the proteolytic activity. Analyses using the photoaffinity labeling technique and systematic mutational studies revealed that the phenylimidazole-type GSM targets a previously unidentified extracellular binding pocket within the N-terminal fragment of presenilin (PS). Collectively, we provide a model for the mechanism of action of the phenylimidazole-type GSM in which binding at the luminal side of PS induces a conformational change in the catalytic center of γ-secretase to modulate Aβ production.

Phosphoinositides Suppress γ-Secretase in Both the Detergent-soluble and -insoluble States

γ-Secretase is an aspartic protease that hydrolyzes type I membrane proteins within the hydrophobic environment of the lipid bilayer. Using the CHAPSO-solubilized γ-secretase assay system, we previously found that γ-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 γ-secretase. Recently, level of secreted β-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 γ-secretase activity. In this study, we found that phosphoinositides directly inhibited CHAPSO-solubilized γ-secretase activity. Interestingly, neither phosphatidylinositol nor inositol triphosphate altered γ-secretase activity. PI(4,5)P2 was also found to inhibit γ-secretase activity in CHAPSO-insoluble membrane microdomains (rafts). Kinetic analysis of β-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, γ-secretase is enriched in rafts. Taking these data together, we propose that phosphoinositides potentially regulate γ-secretase activity by suppressing its association with the substrate.

Effect of helical conformation and side chain structure on γ-secretase inhibition by β-peptide foldamers: insight into substrate recognition.

Substrate-selective inhibition or modulation of the activity of γ-secretase, which is responsible for the generation of amyloid-β peptides, might be an effective strategy for prevention and treatment of Alzheimers disease. We have shown that helical β-peptide foldamers are potent and specific inhibitors of γ-secretase. Here we report identification of target site of the foldamers by using a photoaffinity probe. The photoprobe directly and specifically labeled the N-terminal fragment of presenilin 1, in which the initial substrate docking site is predicted to be located. We also optimized the foldamer structure by preparing a variety of derivatives and obtained two highly potent foldamers by incorporation of a hydrophilic and neutral functional group into the parent structure. The class of side chain functional group and the position of incorporation were both important for γ-secretase-inhibitory activity. The substrate selectivity of the inhibitory activity was also quite sensitive to the class of side chain group incorporated.

Single Chain Variable Fragment against Nicastrin Inhibits the γ-Secretase Activity

γ-Secretase is a membrane protein complex that catalyzes intramembrane proteolysis of a variety of substrates including the amyloid β precursor protein of Alzheimer disease. Nicastrin (NCT), a single-pass membrane glycoprotein that harbors a large extracellular domain, is an essential component of the γ-secretase complex. Here we report that overexpression of a single chain variable fragment (scFv) against NCT as an intrabody suppressed the γ-secretase activity. Biochemical analyses revealed that the scFv disrupted the proper folding and the appropriate glycosyl maturation of the endogenous NCT, which are required for the stability of the γ-secretase complex and the intrinsic proteolytic activity, respectively, implicating the dual role of NCT in the γ-secretase complex. Our results also highlight the importance of the calnexin cycle in the functional maturation of the γ-secretase complex. The engineered intrabodies may serve as rationally designed, molecular targeting tools for the discovery of novel actions of the membrane proteins.

Cooperative Roles of Hydrophilic Loop 1 and the C-Terminus of Presenilin 1 in the Substrate-Gating Mechanism of γ-Secretase

γ-Secretase is a multisubunit protease complex that is responsible for generating amyloid-β peptides, which are associated with Alzheimer disease. The catalytic subunit of γ-secretase is presenilin 1 (PS1), which contains an initial substrate-binding site that is distinct from the catalytic site. Processive cleavage is suggested in the intramembrane-cleaving mechanism of γ-secretase. However, it largely remains unknown as to how γ-secretase recognizes its substrate during proteolysis. Here, we identified that the α-helical structural region of hydrophilic loop 1 (HL1) and the C-terminal region of human PS1 are distinct substrate-binding sites. Mutational analyses revealed that substrate binding to the HL1 region is critical for both ε- and γ-cleavage, whereas binding to the C-terminal region hampers γ-cleavage. Moreover, we propose that substrate binding triggers conformational changes in PS1, rendering it suitable for catalysis. Our data provide new insights into the complicated catalytic mechanism of PS1.