Abstract
The γ-secretase complex cleaves the carboxy-terminal 99 residue (C99) fragment of the amyloid precursor protein (APP) to generate the amyloid-β (Aβ) peptide. The catalytic activity of this complex is mediated either by the presenilin-1 (PS1) or the presenilin-2 (PS2) subunit. In vitro and in vivo studies have demonstrated that PS1-containing complexes generate more total Aβ product than PS2-containing complexes, indicating greater cleavage activity by PS1-containing γ-secretase complexes at the APP γ-site. However, it remains untested whether γ-secretase cleavage at the APP ε-site, which precedes γ-site cleavage and produces the physiologically active APP intracellular domain (AICD), follows the same rule. Using a novel Swedish APP-GVP substrate to facilitate the parallel detection of Aβ and AICD products from PS1-/-/PS2-/- cells co-transfected with either PS1 or PS2, we observed that while PS1 generates more total Aβ product than PS2, consistent with published reports, PS1 and PS2 unexpectedly generate equal amounts of AICD product. We also observed that PS1 and PS2 produce equivalent amounts of Notch intracellular domain (NICD), indicating equal cleavage activity at the Notch S3-site (the corollary of the APP ε-site). Our findings suggest that processivity differences between PS1 and PS2 underlie the differential production of Aβ peptide. Taken together these findings offer novel insights into γ-secretase biology and have important implications for therapeutically targeting γ-secretase
Highlights
-Secretase is an integral membrane protease that mediates the intra-membrane cleavage of over 60 known substrates involved in a range of physiological processes [1,2,3]
Together these findings indicate that PS1 and PS2 generate different amounts of A product yet equal amounts of APP intracellular domain (AICD)-GVP product
We present direct evidence that PS1 and PS2 generate equal amounts of AICD product (Fig. 1C, 1D, 1E, 1F) from an Amyloid precursor protein (APP) fusion protein substrate, as well as equal amounts of Notch intracellular domain (NICD) product (Fig. 2B, 2D, 2E) from two constitutive Notch substrates
Summary
-Secretase is an integral membrane protease that mediates the intra-membrane cleavage of over 60 known substrates involved in a range of physiological processes [1,2,3]. With the exception of one report [10], little is known about the relative activity of PS1- and PS2-containing -secretase complexes in the initial APP substrate -site cleavage event leading to AICD release. In this study we introduced an APP-GVP fusion substrate into a PS1-/-/PS2-/- fibroblast line and, by co-transfecting either PS1 or PS2 to transiently reconstitute -secretase activity, provide direct evidence that PS1 and PS2 generate equal levels of AICD product. By extension, this finding indicates that PS1 and PS2 yield different AICD:A product ratios since it is well documented (and we show in our system) that PS1 generates more total A product than PS2.
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