The Transmembrane Domain Region of Nicastrin Mediates Direct Interactions with APH-1 and the γ-Secretase Complex

Abstract

Nicastrin (NCT) is a type I integral membrane protein that is one of the four essential components of the ␥-secretase complex, a protein assembly that catalyzes the intramembranous cleavage of the amyloid precursor protein and Notch. Other ␥-secretase components include presenilin-1 (PS1), APH-1, and PEN-2, all of which span the membrane multiple times. The mechanism by which NCT associates with the ␥-secretase complex and regulates its activity is unclear. To avoid the misfolding phenotype often associated with introducing deletions or mutations into heavily glycosylated and disulfidebonded proteins such as NCT, we produced chimeras between human (hNCT) and Caenorhabditis elegans NCT (ceNCT). Although ceNCT did not associate with human ␥-secretase components, all of the ceNCT/hNCT chimeras interacted with ␥-secretase components from human, C. elegans, or both, indicating that they folded correctly. A region at the C-terminal end of hNCT, encompassing the last 50 residues of its ectodomain, the transmembrane domain, and the cytoplasmic domain was important for mediating interactions with human PS1, APH-1, and PEN-2. This finding is consistent with the fact that the bulk of the ␥-secretase complex proteins resides within the membrane, with relatively small extramembranous domains. Finally, hNCT associated with hAPH-1 in the absence of PS, consistent with NCT and APH-1 forming a subcomplex prior to association with PS1 and PEN-2 and indicating that the interactions between NCT with PS1 may be indirect or stabilized by the presence of APH-1.

Requirement of PEN-2 for Stabilization of the Presenilin N-/C-terminal Fragment Heterodimer within the γ-Secretase Complex

“…Interestingly, immature NCT and APH-1aL as well as the low levels of residual mature NCT (because of the incomplete PEN-2 knockdown) remained stable (Fig. 2B), consistent with the observation that these two proteins can independently stabilize each other (13,20,24,25). Taken together, these data demonstrate that PEN-2-⌬C is highly unstable.…”

“…This is in contrast to correctly assembled ␥-secretase complex components, which have been shown to be very stable and have a long half-life time (9,17,44,48). The failure of PEN-2-⌬C to stably assemble with PS, NCT, and APH-1 was associated with a marked selective instability of the PS1 NTF and CTF, which also underwent proteasomal degradation, whereas NCT and APH-1 were stable, consistent with previous findings that these proteins can stabilize each other independently (20,24,25). These data suggest that PEN-2 has a dual function as follows: a function in the initiation of PS endoproteolysis as a first maturation step during ␥-secretase complex assembly, and a second function in the stabilization of the PS fragment heterodimer within the ␥-secretase complex that is required for further maturation of the ␥-secretase complex.…”

“…4). The anti-NCT antibody coimmunoprecipitated APH-1aL with immature NCT in the CHAPSO-solubilized membrane fractions from the PEN-2-⌬C-expressing cells, consistent with their independent stable interaction (13,20,24,25). Thus, these data demonstrate that PEN-2-⌬C does not undergo stable ␥-secretase complex formation.…”

“…Subsequent assembly of PEN-2 with this intermediate drives the conversion of the PS holoprotein into the active heterodimer (17). Furthermore, a potential NCT/APH-1 assembly intermediate has been observed recently (24), and NCT has been shown to stably interact with APH-1 in the absence of PS (20,25). Interestingly, ␥-secretase complex assembly is associated with a conformational change of the NCT ectodomain, which adopts a protease-resistant conformation (26).…”

Characterization of presenilin complexes from mouse and human brain using Blue Native gel electrophoresis reveals high expression in embryonic brain and minimal change in complex mobility with pathogenic presenilin mutations

“…Evidence for selfassociation of APH-1a with itself and with APH-1b was also reported previously [16]. Morais et al demonstrated that NCT and APH-1 were able to interact in vitro in the absence of PS [44]. Our results suggest that all four putative complex components could be detected in the PS complex for embryonic mouse brain after BN/PAGE, and that APH-1 association may be diminished by carbonate wash.…”