PEN‐2 enhances γ‐cleavage after presenilin heterodimer formation

Abstract

The presenilin (PS) complex, including PS, nicastrin, APH-1 and PEN-2, is essential for c-secretase activity, which is required for amyloid b-protein (Ab) generation. However, the precise individual roles of the three cofactors in the PS complex in Ab generation remain to be clarified. Here, to distinguish the roles of PS cofactors in c-secretase activity from those in PS endoproteolysis, we investigated their roles in the c-secretase activity reconstituted by the coexpression of PS N-and C-terminal fragments (NTF and CTF) in PS-null cells. We demonstrate that the coexpression of PS1 NTF and CTF forms the heterodimer and restores Ab generation in PS-null cells. The generation of Ab was saturable at a certain expression level of PS1 NTF/CTF, while the overexpression of PEN-2 alone resulted in a further increase in Ab generation. Although PEN-2 did not enhance PS1 NTF/CTF heterodimer formation, PEN-2 expression reduced the IC 50 of a specific c-secretase inhibitor, a transition state analogue, for Ab generation, suggesting that PEN-2 expression enhances the affinity or the accessibility of the substrate to the catalytic site. Thus, our results strongly suggest that PEN-2 is not only an essential component of the c-secretase complex but also an enhancer of c-cleavage after PS heterodimer formation.

Glu-333 of Nicastrin Directly Participates in γ-Secretase Activity

“…Indeed, recent data suggests that HDVII adopts distinct conformations with PS holoprotein and endoproteolyzed PS (31). ␥-Secretase activation may include rearrangement of the PS catalytic aspartate residues upon binding of PEN-2 to the complex, which is concomitant with PS endoproteolysis (32)(33)(34)(35)(36). Once the active site has been established, HDVII is cleaved and vacates the active site to allow for entry of substrates.…”

Aβ42 Overproduction Associated with Structural Changes in the Catalytic Pore of γ-Secretase

“…Full-length cDNAs encoding APP carrying Swedish mutation (APP NL ), wild-type human PS1, and single-Cys mutants (I114C, A246C, or L250C) based on cysteine-less PS1 in pLPCX (Clontech) or pMX-puro (provided from Dr. T. Kitamura) were described (8). Human NCT (hNCT), APH-1b (hAPH-1b), and untagged and FLAGtagged hPEN-2 in pMX vector were kindly provided by Dr. Komano (20). hPEN-2/⌬2-10 in pMX was generated by long-PCR-based mutagenesis.…”

“…B, effect of the N-terminally tag of dPen-2 on relative luciferase activity in S2 reporter cell line transiently transfected with dPen-2, Psn, dNct-V5/His, and dAph-1-FLAG (n ϭ 12, mean Ϯ S.E.). obtained from Psen1/Psen2 double knock-out mice (DKO cells) stably expressing APP Swedish mutant (20). Generation of A␤42 from cells expressing FLAG-hPEN-2 was dramatically augmented at 1.9-fold compared with that from untagged hPEN-2-expressing cells (Table 5).…”

Angiotensin-Converting Enzyme Converts Amyloid β-Protein 1–42 (Aβ1–42) to Aβ1–40, and Its Inhibition Enhances Brain Aβ Deposition

“…A␤ 1-42 and A␤ 1-40 levels were determined using the ELISA kit. Fibroblasts stably expressing human APP695 (Shiraishi et al, 2004) were grown in DMEM (Invitrogen, Grand Island, NY) containing 10% fetal calf serum. The medium was changed at 100% confluence, and the fibroblasts were treated with or without captopril.…”