| Autor: |
Tomasselli, Alfredo G., Qahwash, Isam, Emmons, Thomas L., Lu, Yifeng, Leone, Joseph W., Lull, June M., Fok, Kam F., Bannow, Carol A., Smith, Clark W., Bienkowski, Michael J., Heinrikson, Robert L., Yan, Riqiang |
| Předmět: |
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| Zdroj: |
Journal of Neurochemistry; 3/1/2003, Vol. 84 Issue 5, p1006-1017, 12p |
| Abstrakt: |
Abstract The involvement of β-secretase (BACE1; β-site APP-cleaving enzyme) in producing the β-amyloid component of plaques found in the brains of Alzheimer's patients, has fueled a major research effort to characterize this protease. Here, we describe work toward understanding the substrate specificity of BACE1 that began by considering the natural APP substrate and its Swedish mutant, APP[sub Sw], and proceeded on to include oxidized insulin B chain and ubiquitin substrates. From these findings, and the study of additional synthetic peptides, we determined that a decapeptide derived from APP in which the P3-P2′ sequence, ...VKM-↓-DA..., was replaced by ...ISY-↓-EV... (-↓- = β site of cleavage), yielded a substrate that was cleaved by BACE1 seven times faster than the corresponding APP[sub Sw] peptide, SEVNL-↓-DAEFR. The expanded peptide, GLTNIKTEEISEISY-↓-EVEFRWKK, was cleaved an additional seven times faster than its decapeptide counterpart (boldface), and provides a substrate allowing assay of BACE1 at picomolar concentrations. Several APP mutants reflecting these β-site amino acid changes were prepared as the basis for cellular assays. The APP[sub ISYEV] mutant proved to be a cellular substrate that was superior to APP[sub Sw]. The assay based on APP[sub ISYEV] is highly specific for measuring BACE1 activity in cells; its homolog, BACE2, barely cleaved APP[sub ISYEV] at the β-site. Insertion of the optimized ISY-↓-EV motif at either the β-site (Asp1) or β′-site (Glu11) directs the rate of cellular processing of APP at these two accessible sites. Thus, we have identified optimal BACE1 substrates that will be useful to elucidate the cellular enzymatic actions of BACE1, and for design of inhibitors that might be of therapeutic benefit in Alzheimer's disease. [ABSTRACT FROM AUTHOR] |
| Databáze: |
Complementary Index |
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