Reconstitution of γ‐secretase by truncated presenilin (PS) fragments revealed that PS C‐terminal transmembrane domain is critical for formation of γ‐secretase complex

Abstract

The presenilin (PS) complex, including PS, nicastrin (NCT), APH-1 and PEN-2, is essential for γ γ γ γ -secretase activity. Previously, the PS C-terminal tail was shown to be essential for γ γ γ γ -secretase activity. Here, to further understand the precise mechanism underlying the activation of γ γ γ γ -secretase regulated by PS cofactors, we focused on the role of the PS1 C-terminal region including transmembrane domain (TM) 8 in γ γ γ γ -secretase activity. For this purpose, we co-expressed Cterminally truncated PS1 (PS1∆ ∆ ∆ ∆ C) completely lacking γ γ γ γ -secretase activity and the PS1 C-terminal short fragment in PS-null cells, because the successful reconstitution of γ γ γ γ -secretase activity in PSnull cells by the co-expression of PS1∆ ∆ ∆ ∆ C and the PS1 C-terminal short fragment would allow us to investigate the role of the PS1 C-terminal region in γ γ γ γ -secretase activity. We found that the exogenous expression of the PS1 C-terminal short fragment with NCT and APH-1 completely rescued a defect of the γ γ γ γ -secretase activity of PS1∆ ∆ ∆ ∆ C in PS-null cells. With this reconstitution system, we demonstrate that both TM8 and the PS1 C-terminal seven-amino-acid-residue tail are involved in the formation of the active γ γ γ γ -secretase complex via the assembly of PS1 with NCT and APH-1.

γ-Secretase and Presenilin Mediate Cleavage and Phosphorylation of Vascular Endothelial Growth Factor Receptor-1

“…In the case of APH-1, our siRNA targeted APH-1αL because it has been suggested to be the dominant form of APH-1. However, recent studies report that multiple isoforms (αL, αS, and β) of APH-1 can replace each other in the γ-secretase complex (35, 36), and this may provide an explanation why our siRNA against APH-1 has less of an effect on γ-secretase activity. It has been shown that PS1 knockdown leads to a strong reduction of PEN-2 and that the PEN-2 level is also down-regulated upon siRNA knockdown of nicastrin (37).…”

Presenilin Transmembrane Domain 8 Conserved AXXXAXXXG Motifs Are Required for the Activity of the γ-Secretase Complex

“…Co-immunoprecipitation-Co-immunoprecipitation was carried out based on Shiraishi et al (44) and Xia et al (55). 48 h after transfection, CHO cells were washed with cold PBS and homogenized in lysis buffer A (50 mM Tris, pH 7.6, 150 mM NaCl, 2 mM EDTA, 1% CHAPS, and protease inhibitor mixture).…”

“…This domain is involved in the formation and stability of the complex (44,45) and in the membrane integration of TMD7 into the membrane bilayer by "forced transmembrane orientation" (43). In PS1, both motifs include two critical amino acids: Gly-417, having a pivotal role in ␥-secretase activity (46), and Ala-409, whose substitution with Thr (PS1 A409T) is described as a FAD mutation (47).…”

Cholesterol‐induced astrocyte activation is associated with increased amyloid precursor protein expression and processing

“…In healthy individuals, most of the APP is constitutively cleaved within the Aβ sequence by α‐secretase enzymes (TACE/ADAM17 and/or ADAM10) generating the soluble ectodomain of APP (sAPPα) and a truncated C‐terminal peptide (CTF‐α or C‐83). In the amyloidogenic pathway, APP is proteolyzed by β‐secretase (BACE‐1) forming the smaller membrane bound C‐terminal fragment (CTF‐β or C‐99), which is further cleaved by the γ‐secretase leading to Aβ formation (Cai et al, ; De Strooper, ; De Strooper and Annaert, ; Glenner and Wong, ; Iwatsubo, ; Shiraishi et al, ; Vassar et al, ).…”