Amyloidogenic Processing but Not Amyloid Precursor Protein (APP) Intracellular C-terminal Domain Production Requires a Precisely Oriented APP Dimer Assembled by Transmembrane GXXXG Motifs

Abstract

The ␤-amyloid peptide (A␤) is the major constituent of the amyloid core of senile plaques found in the brain of patients with Alzheimer disease. A␤ is produced by the sequential cleavage of the amyloid precursor protein (APP) by ␤-and ␥-secretases. Cleavage of APP by ␥-secretase also generates the APP intracellular C-terminal domain (AICD) peptide, which might be involved in regulation of gene transcription. APP contains three Gly-XXX-Gly (GXXXG) motifs in its juxtamembrane and transmembrane (TM) regions. Such motifs are known to promote dimerization via close apposition of TM sequences. We demonstrate that pairwise replacement of glycines by leucines or isoleucines, but not alanines, in a GXXXG motif led to a drastic reduction of A␤40 and A␤42 secretion. ␤-Cleavage of mutant APP was not inhibited, and reduction of A␤ secretion resulted from inhibition of ␥-cleavage. It was anticipated that decreased ␥-cleavage of mutant APP would result from inhibition of its dimerization. Surprisingly, mutations of the GXXXG motif actually enhanced dimerization of the APP C-terminal fragments, possibly via a different TM ␣-helical interface. Increased dimerization of the TM APP C-terminal domain did not affect AICD production.The progressive deposition of ␤-amyloid peptide (A␤) 3 leading to the formation of senile plaques is an invariant feature of Alzheimer disease. A␤ is a 39 -43-amino acid peptide, with two major isoforms of 40 and 42 amino acids (1, 2). A␤ is produced by the amyloidogenic cleavage of its precursor, the amyloid precursor protein or APP (3).The amyloidogenic processing of APP is initiated by ␤-cleavage within the lumenal/extracellular domain of the protein.The ␤-cleavage of APP is performed by the BACE proteins (BACE1 and -2) that are integral membrane proteins belonging to the aspartyl protease family (4 -8). ␤-Cleavage produces a 99-amino acid, membrane-anchored APP C-terminal fragment (␤CTF), which is further cleaved by the ␥-secretase activity to generate A␤. The ␥-secretase activity is contained in a high molecular weight multiprotein complex formed at least by the following proteins: a presenilin (PS1 or PS2), nicastrin (Nct), Pen-2, and Aph-1 (9). The activity of the ␥-secretase complex is also required for the generation of the intracellular fragment named (APP intracellular C-terminal domain (AICD). AICD was shown to translocate to the nucleus (10, 11), and there is growing experimental evidence suggesting a role for AICD in the regulation of gene transcription (12-17) even if the identity of APP target genes remains a matter of debate (18). The ␥-secretase complex, therefore, plays a central role in the onset and progression of Alzheimer disease not only because proteolysis of ␤CTF controls the production of A␤, but it also controls the intracellular signaling associated with APP, which in turn might regulate the expression of genes involved in the disease. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "ad...

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

“…Taken together, these results have led to the idea that APP dimerization can positively regulate A␤ production. However, other laboratories have not been able to confirm some of these observations using slightly different approaches (23,27 …”

“…In this model these processive cleavages can be perturbed while keeping ⑀-cleavage relatively intact. In fact, a recent publication showed that mutations within the GXXXG motif in the APP transmembrane domain led to a reduction in A␤40 and A␤42 without altering AICD levels (23). In this latter case, however, the drop in A␤40 and A␤42 levels was accompanied by an increase in A␤34 species (21), indicating that mutations at the GXXXG motif were specific for the generation of A␤ peptides, presumably due to alterations to ␥-secretase cleavage.…”

“…In addition, based primarily on artificial cysteine mutations designed to induce disulfide bond formation, it has been suggested that APP dimerization accelerates A␤ production (25). However, subsequent studies have not consistently confirmed these initial observations (23,27). In this study we chose to use a different approach to induce APP dimerization to explore the relationship between dimerization and A␤ generation without having to introduce cysteine mutations by creating APP-FKBP chimeric proteins.…”

“…Finally, there are other examples wherein A␤ and AICD production are not linked. For example, a number of mutations within the APP transmembrane region were shown to have distinct effects on the two cleavage events (23,49,50). And, TMP21, a member of the p24 cargo protein family, appeared to selectively inhibit APP processing at the ␥-site without affecting ⑀-cleavage (51).…”

“…To date at least four domains have been reported to promote APP dimerization; that is, the E1 domain containing the N-terminal growth factor-like domain and copper binding domain (17), the E2 domain containing the carbohydrate domain in the APP ectodomain (19), the APP juxtamembrane region (20), and the transmembrane domain (21,22). In the latter domain the dimerization appears to be mediated by the GXXXG motif near the luminal face of the transmembrane region (21,23). In addition to promoting cell adhesion, APP dimerization has been proposed to increase susceptibility to cell death (20,24).…”