RESUMEN
Presenilin (PS1 or PS2) is an essential component of the active gamma-secretase complex that liberates the Abeta peptides from amyloid precursor protein (APP). PS1 is regarded as an atypical aspartyl protease harboring two essential aspartic acids in the context of the sequence D257LV and D385FI, respectively, rather than the typical DTG...DTG catalytic motif of classical aspartyl proteases. In the present studies, we introduced the sequence DTG in PS1 at and around the catalytic D257 and D385 residues to generate three PS1 mutants: D257TG, D385TG, and the double-mutant D257TG/D385TG. The effects of these changes on the gamma-secretase activity in the presence or absence of gamma-secretase inhibitors and modulators were investigated. The results showed that PS1 mutants having D385TG robustly enhanced Abeta42 production compared to the wild type (wt), and were more sensitive than wt to inhibition by a classical aspartyl protease transition state mimic, and fenchylamine, a sulfonamide derivative. Unlike wt PS1 and some of its clinical mutants, all three PS1 artificial mutants decreased cleavage of Notch S3-site, suggesting that these artificial mutations may trigger conformational changes at the substrate docking and catalytic site that cause alteration of substrate specificity and inhibition pattern. Consistent with this notion, we have found that NSAID enzymatic inhibitors of COX, known modulators of the gamma-secretase activity, cause PS1 mutants containing D385TG to produce higher levels of both Abeta38 and Abeta42, but to reduce levels of Abeta39, showing a pattern of Abeta formation different from that observed with wild type PS1 and its clinical mutants. This study provides an important structural clue for the rational design of drugs to inhibit processing of APP at the gamma-site without interfering with Notch processing.
RESUMEN
Brain plaque deposits of amyloid-beta peptide (Abeta) is a pathological hallmark of Alzheimer's disease (AD) and apolipoprotein E (apoE) is thought to be involved in its deposition. One hypothesis for the role of apoE in the pathogenesis of AD is that apoE may be involved in deposition or clearance of Abeta by direct protein-to-protein interaction. Lipidated apoE4 bound preferentially to an intermediate aggregated form of Abeta and formed two- to three-fold more binding complexes than isoforms apoE2 or apoE3. The interaction was detected by a sandwich ELISA with capture antibodies specific for the N-terminus of apoE, whereas the interaction was not recognized with a C-terminal antibody. The observations indicate that the C-terminus of apoE4 interacts with the intermediate form of Abeta. The differential risk of AD related to apoE genotype may be the result of an enhanced capacity of apoE4 binding to an intermediate aggregated form of Abeta.
Asunto(s)
Péptidos beta-Amiloides/química , Apolipoproteínas E/química , Fragmentos de Péptidos/química , Apolipoproteína E2 , Apolipoproteína E3 , Apolipoproteína E4 , Ensayo de Inmunoadsorción Enzimática , Humanos , Unión Proteica , Isoformas de Proteínas/química , Estructura Terciaria de ProteínaRESUMEN
Brain plaque deposition in the form of amyloid-beta (Abeta) peptide is a pathological hallmark of Alzheimer's disease (AD). Apolipoprotein E (ApoE) is thought to be involved in plaque formation. Individuals afflicted with AD carrying the ApoE4 isoform have shown a greater number of Abeta plaques when compared to ApoE3 carriers, and inheritance of an ApoE4 allele increases the risk of AD when compared to ApoE2 and ApoE3 carriers. The role of ApoE in the pathogenesis of AD is not well understood but a hypothesis gaining widespread support is that ApoE is involved in deposition or clearance of Abeta by direct protein-to-protein interaction. We have established that various human Abeta conformations apparent during spontaneous aggregation confer differing degrees of binding to the three ApoE isoforms. When associated with lipid, ApoE4 bound preferentially to an intermediate aggregated form of Abeta and had higher avidity than did ApoE2 or ApoE3.