Localization of mature neprilysin in lipid rafts.

Alzheimer's disease (AD) is characterized by senile plaques caused by amyloid-ß peptide (Aß) accumulation. It has been reported that Aß generation and accumulation occur in membrane microdomains, called lipid rafts, which are enriched in cholesterol and glycosphingolipids. Moreover, the ablation of cholesterol metabolism has been implicated in AD. Neprilysin (NEP), a neutral endopeptidase, is one of the major Aß-degrading enzymes in the brain. Activation of NEP is a possible therapeutic target. However, it remains unknown whether the activity of NEP is regulated by its association with lipid rafts. Here we show that only the mature form of NEP, which has been glycosylated in the Golgi, exists in lipid rafts, where it is directly associated with phosphatidylserine. Moreover, the localization of NEP in lipid rafts is enhanced by its dimerization, as shown using the NEP E403C homodimerization mutant. However, the protease activities of the mature form of NEP, as assessed by in vitro peptide hydrolysis, did not differ between lipid rafts and nonlipid rafts. We conclude that cholesterol and other lipids regulate the localization of mature NEP to lipid rafts, where the substrate Aß accumulates but does not modulate the protease activity of NEP.

Inhibition by KMI-574 leads to dislocalization of BACE1 from lipid rafts.

BACE1 initiates processing of the amyloid precursor protein (APP) in the production of amyloid beta (Abeta) peptide. After beta-cleavage by BACE1, the C-terminal stub of the APP fragment is further processed by the gamma-secretase complex to produce Abeta. Because APP, Abeta, the gamma-secretase complex, and BACE1 are found in lipid raft membranes, Abeta production is widely accepted to occur in lipid rafts. However, whether BACE1 is activated within the rafts is unclear. To analyze the relationship between the activity and the localization of BACE1, we used a new BACE1 inhibitor, KMI-574, and separated raft membranes on sucrose density gradients. In the presence of KMI-574, the localization of BACE1 shifted from the rafts to nonraft membranes in HEK293 cells. We also analyzed the proteolytically inactive mutants, D93A, D289A, and D93A/D289A, of BACE1. These mutants also moved from rafts to nonrafts, and the D93A/D289A double-mutant localized exclusively to nonraft membranes. The mutants were defective in maturation by glynosylation and formed hyperoligomers, suggesting that the BACE1 oligomers could not exit from the ER and be transported to the Golgi apparatus. Our findings suggest that the activated conformation of BACE1 is important for protein transport and localization to lipid rafts.

1,3-Capryloyl-2-arachidonoyl glycerol activates alpha-secretase activity and suppresses Abeta40 secretion in A172 cells.

Alzheimer's disease (AD) is characterized by the deposition of amyloid beta-peptide (Abeta), derived from amyloid precursor protein (APP). Membrane states, such as lipid components or membrane fluidity, are important for enzymes related to APP processing in meeting their substrates efficiently. We analyzed the effects of triglycerides combined with polyunsaturated fatty acids (PUFAs) and/or caprylic acids on APP proteolysis. Our results showed that 1,3-capryloyl-2-arachidonoyl glycerol (8A8) moderately increased alpha-secretase activity (18%) in A172 cells. beta-Secretase activity was not statistically significantly changed in HEK293 cells stably expressing BACE1. However, Abeta40 secretion decreased by 22%. Thus, we conclude that 8A8 is a useful lipid compound for activating alpha-secretase activity and suppressing Abeta40 secretion.

beta-Secretase inhibitors: modification at the P4 position and improvement of inhibitory activity in cultured cells.

Recently, we reported potent and small-sized beta-secretase (BACE1) inhibitors KMI-570 and KMI-684 in which we replaced carboxylic acid groups at the P(1)(') position of KMI-420 and KMI-429, respectively, with tetrazole derivatives as carboxylic acid bioisosteres. These modifications improved significantly BACE1 inhibitory activity and chemical stability. In this study, the acidic tetrazole ring of the P(4) position of KMI-420 and KMI-570, respectively, was replaced with various hydrogen bond acceptor groups. We found BACE1 inhibitor KMI-574 that exhibited potent inhibitory activity in cultured cells as well as in vitro enzymatic assay.