Distinct Intramembrane Cleavage of the β-Amyloid Precursor Protein Family Resembling γ-Secretase-like Cleavage of Notch

Abstract

The intramembrane cleavage of ␤-amyloid precursor protein by ␥-secretase is the final step in the generation of amyloid ␤-protein. A 59-or 57-residue C-terminal fragment called CTF␥ is produced concomitantly. Putative CTF␥ generated in rat brain membrane preparations was purified and sequenced. Instead of CTF␥, shorter 50-and 49-residue fragments were identified. In addition, we found similar C-terminal fragments of ␤-amyloid precursor-like proteins 1 and 2; these were also cleaved at corresponding sites. This newly identified cleavage occurs at a site two to five residues inside the cytoplasmic membrane boundary, which is very similar to ␥-secretase-like cleavage of Notch 1. Cerebral deposition of amyloid ␤-protein (A␤)1 is an early, invariant, and essential event in the pathogenesis of Alzheimer's disease (AD) (1). A␤ is generated from ␤-amyloid precursor protein (APP) when ␤-secretase cleavage at the extracellular domain produces a 99-residue C-terminal fragment called CTF99 or CTF␤. Subsequent cleavage of CTF␤ in the middle of the transmembrane domain by ␥-secretase primarily produces either a 40-residue protein (A␤40) or a 42-residue protein (A␤42). A␤42 has the higher aggregation potential and is the first species to accumulate in aged and AD brains (2-4). Intramembrane ␥-secretase cleavage of CTF␤ should yield a 59-or 57-residue cytoplasmic C-terminal fragment (CTF41-99 or CTF43-99; APP712-770 or APP714 -770 according to the numbering of the APP770 isoform) called CTF␥. However, CTF␥has not yet been identified in either brain homogenates or cell lysates, presumably because of its instability in vivo. To learn more about the properties of ␥-cleavage, we decided to characterize CTF␥. EXPERIMENTAL PROCEDURES Subcellular Fractionation of Newborn Rat Brains and Chinese Hamster Ovary (CHO) CellsStably Expressing APP751-Fresh newborn (day 1) rat brains were homogenized on ice with 20 strokes in a glassTeflon homogenizer in eight volumes of Buffer H (20 mM Hepes, 150 mM NaCl, 10% glycerol, 5 mM EDTA, pH 7.4). All subsequent steps were carried out at 4°C unless indicated otherwise. The nuclear fractions were collected by centrifugation of the homogenates at 2,500 ϫ g (brains) or at 800 ϫ g (CHO cells) for 10 min, followed by a brief washing in Buffer H with or without 0.4% Triton X-100. The postnuclear supernatant was separated into cytosolic and membranous fractions by centrifugation at 100,000 ϫ g for 1 h. Each fraction was adjusted to an equal volume for Western blotting.Preparation of Active Membrane Fractions-The postnuclear supernatant from newborn rat brains, as prepared above, was spun at 10,000 ϫ g for 15 min to collect membrane fraction F2P, with additional subsequent spin at 100,000 ϫ g for 1 h to collect F3P. F2P was collected from the CHO cells by centrifugation at 100,000 ϫ g for 1 h. The membrane fractions, after being washed once with the buffer, were incubated at 37°C in Buffer H containing a mixture of protease inhibitors (5 mM EDTA, 5 mM EGTA, 5 mM 1,10-phenanthroline, 10 M bestatin, 10 M amasta...

Intracellular domains of amyloid precursor-like protein 2 interact with CP2 transcription factor in the nucleus and induce glycogen synthase kinase-3β expression

“…8,10 The APLP family can be also cleaved by esecretase to generate the ICD composed of the last 50 amino acids of APLPs C-terminus (C50). 13,14 Moreover, the ICDs of APLP1 and APLP2 produced by g-secretase enhanced Fe65-dependent gene transcriptional activation, as have been reported for the APP intracellular domain (AICD). 10 Fe65, which is known to be ones of the adaptor proteins for APP, contains three protein-protein interaction domains, a WW and two phosphotyrosine binding (PTB) domains.…”

“…26,27 APP, which is a type I transmembrane protein as well as APLPs, is cleaved consecutively, first at the extracellular juxtamembrane region by a-or b-secretase and secondly at the intramembrane region by g-secretase. 26 Similarly with APP, the N-terminal ectodomain of APLP2 is shedded by b-secretase 28,29 as well as by ADAM 10 and 17, 30 whereas the C-termini remain in the membrane 10,28 and can be further processed by g-or e-secretases 13,14 to release ICDs with signaling properties. 10,28 The APLP2-ICDs are known to interact with Fe65 via a YENPTY domain, which APP, APLP1 and APLP2 have in common in their C-terminus, translocating into the nucleus by Fe65-dependent manner 8 and show Fe65-dependent gene transcriptional activity by heterologous reporter genes.…”

Syndecan 3 Intramembrane Proteolysis Is Presenilin/γ-Secretase-dependent and Modulates Cytosolic Signaling

“…This was not entirely unexpected, because, as for other substrates ␥-secretase, it is extremely difficult to detect ICD, probably because of rapid intracellular turnover (31,32). Yet, several groups have developed cell-free assays, allowing them to demonstrate ICD generation from APP by ␥-secretase (28,(33)(34)(35)(36). Using a similar assay for syndecan, we detected a 7-kDa fragment that was continuously released over a period of at least 4 h into the supernatant of S3-MEF membranes (Fig.…”

“…A V1744G mutation at P1Ј has been shown to strongly interfere with ␥-secretase processing of Notch (38), whereas a V646G mutation at P1Ј of APP is without effect (28,33,34). Surprisingly, the syndecan V404G mutation caused a marked increase in ␥-secretase cleavage, as judged by an increased ratio of SICD to CTF (Fig.…”

Trafficking and Proteolytic Processing of APP

“…The subsequent intramembrane cut by g-secretase liberates a truncated Ab peptide called p3 (Haass et al 1993b), which apparently is pathologically irrelevant. g-Secretase not only liberates Ab (from C99) and p3 (from C83) but also generates the APP intracellular domain (AICD) (Gu et al 2001;Sastre et al 2001;Weidemann et al 2002), which is released into the cytosol and which may have a function in nuclear signaling (Cao and Sudhof 2001;von Rotz et al 2004). The amyloidogenic and the anti-amyloidogenic processing pathways compete with each other at least in some subcellular loci, since enhancing a-secretase activity in animal models of Alzheimer disease (AD) or in cultured cells can significantly lower Ab generation and even amyloid plaque formation (Nitsch et al 1992;Postina et al 2004).…”