Amyloid β-Protein Precursor Juxtamembrane Domain Regulates Specificity of γ-Secretase-dependent Cleavages

Abstract

Amyloid ␤-protein (A␤), the major component of cerebral plaques associated with Alzheimer disease, is derived from amyloid ␤-protein precursor (APP) through sequential proteolytic cleavage involving ␤-and ␥-secretase. The intramembrane cleavage of APP by ␥-secretase occurs at two major sites, ␥ and ⑀, although the temporal and/or mechanistic relationships between these cleavages remain unknown. In our attempt to address this issue, we uncovered an important regulatory role for the APP luminal juxtamembrane domain. We demonstrated in cell-based assays that domain replacements in this region can greatly reduce secreted A␤ resulting from ␥-cleavage without affecting the ⑀-cleavage product. This A␤ reduction is likely due to impaired proteolysis at the ␥-cleavage site. Further analyses with site-directed mutagenesis identified two juxtamembrane residues, Lys-28 and Ser-26 (A␤ numbering), as the critical determinants for efficient intramembrane proteolysis at the ␥-site. Consistent with the growing evidence that ⑀-cleavage of APP precedes ␥-processing, longer A␤ species derived from the ␥-cleavage-deficient substrates were detected intracellularly. These results indicate that the luminal juxtamembrane region of APP is an important regulatory domain that modulates ␥-secretase-dependent intramembrane proteolysis, particularly in differentiating ␥-and ⑀-cleavages.

Evidence that Enzyme Processivity Mediates Differential Aβ Production by PS1 and PS2

“…To verify that the AICD-GVP product levels measured here did not simply result from some artifact unique to the novel Swedish APP-GVP substrate, we also tested the previously described APP C99-GVP substrate [28]. Similar results were obtained with APP C99-GVP substrate (data not shown), indicating that different APP substrates yield the same result.…”

“…Full-length Swedish APP-GVP was subcloned using the APP C99-GVP backbone (previously reported [28]). Briefly, the ectodomain of Swedish APP695 was PCR amplified and blunt-end ligated in-frame into linearized APP C99-GVP using the In-Fusion PCR Cloning System (Clontech).…”

“…NotchΔE substrate (2 μg) was co-transfected with CBF- Luciferase reporter gene (1 μg) [29, 34] and Swedish APP-GVP substrate (2 μg) was co-transfected with UAS- Luciferase reporter gene (1 μg) [28]. The β-Galactosidase expression plasmid pCMV-LacZ (1 μg, Life Technologies) was also co-transfected with each condition to normalize for transfection efficiency.…”

“…Aβ1-x was quantified using antibody 266 (epitope Aβ16-23) for capture and biotinylated antibody 2H3 (epitope Aβ4-7) for detection, and both antibodies have been described previously [28]. NICD was quantified using antibody 9F3 (N-terminal neo-epitope, [31]) for capture and a biotinylated anti-HA antibody for detection.…”

Induced Dimerization of the Amyloid Precursor Protein Leads to Decreased Amyloid-β Protein Production

“…K28C mutation (using A␤ numbering) showed 6 -7-fold augmentation in A␤ secretion (25), whereas the L17C mutation (21) showed unchanged A␤40 levels but increased A␤42 release, suggesting that dimers of APP facilitate A␤ generation. However, a recent study reported that a K28S mutation showed a marked (90%) reduction in A␤ production (35), possibly due to impairment in ␥-secretase cleavage. As a result, we wish to first revisit these cysteine mutants as well as determine the effects of base substitutions at the lysine residue at position 28 (by A␤ numbering or position 624 by APP695 numbering).…”