Abstract
Amyloid beta-protein (Abeta), the major component of cerebral plaques associated with Alzheimer disease, is derived from amyloid beta-protein precursor (APP) through sequential proteolytic cleavage involving beta- and gamma-secretase. The intramembrane cleavage of APP by gamma-secretase occurs at two major sites, gamma and epsilon, 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 Abeta resulting from gamma-cleavage without affecting the epsilon-cleavage product. This Abeta reduction is likely due to impaired proteolysis at the gamma-cleavage site. Further analyses with site-directed mutagenesis identified two juxtamembrane residues, Lys-28 and Ser-26 (Abeta numbering), as the critical determinants for efficient intramembrane proteolysis at the gamma-site. Consistent with the growing evidence that epsilon-cleavage of APP precedes gamma-processing, longer Abeta species derived from the gamma-cleavage-deficient substrates were detected intracellularly. These results indicate that the luminal juxtamembrane region of APP is an important regulatory domain that modulates gamma-secretase-dependent intramembrane proteolysis, particularly in differentiating gamma- and epsilon-cleavages.
Highlights
Fragment known as amyloid -protein precursor (APP) intracellular domain (AICD) [6, 7]
In the present study we examined the significance of APP luminal juxtamembrane domain in ␥-secretase-mediated proteolysis
Cleavage of C99GVP by ␥-secretase releases an A-like peptide with the extra LE residues on its N terminus [30] as well as an APP intracellular domain (AICD)-like fragment (AICD-GVP) that can transactivate a luciferase reporter through its GVP domain (Fig. 1A)
Summary
⑀-cleavages, a potential mechanism that may be exploitable for developing safe ␥-secretase inhibitors for AD therapy. In the present study we examined the significance of APP luminal juxtamembrane domain in ␥-secretase-mediated proteolysis. Through a series of mutagenesis experiments using a C99-derived substrate that contains a Gal4/VP16 (GVP) signaling domain, we were able to evaluate the effects of juxtamembrane mutations on multiple cleavage events within the same substrate. Similar to the wild type C99, C99-GVP is a functional ␥-secretase substrate cleavable at both ␥- and ⑀-sites. Domain swap as well as point mutations in its luminal juxtamembrane domain led to significant reduction in secreted A without affecting AICD production. The divergent effects of these mutations on ␥- and ⑀-cleavages further support a modulatory role for the APP luminal juxtamembrane domain in cleavage specificity in addition to its known function in substrate recognition and recruitment
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