Secretion of the Notch-1 Aβ-like Peptide during Notch Signaling

Abstract

The canonical pathway of Notch signaling is mediated by regulated intramembrane proteolysis (RIP). In the pathway, ligand binding results in sequential proteolysis of the Notch receptor, and presenilin (PS)-dependent intramembrane proteolysis at the interface between the membrane and cytosol liberates the Notch-1 intracellular domain (NICD), a transcription modifier. Because the degradation of the Notch-1 transmembrane domain is thought to require an additional cleavage near the middle of the transmembrane domain, extracellular small peptides (Notch-1 A␤-like peptide (N␤)) should be produced. Here we showed that N␤ species are indeed secreted during the process of Notch signaling. We identified mainly two distinct molecular species of novel N␤, N␤21 and C-terminally elongated N␤25, which were produced in an ϳ5:1 ratio. This process is reminiscent of the production of Alzheimer disease-associated A␤. PS pathogenic mutants increased the production of the longer species of A␤ (A␤42) from ␤-amyloid protein precursor. We revealed that several Alzheimer disease mutants also cause a parallel increase in the secretion of the longer form of N␤. Strikingly, chemicals that modify the A␤42 level caused parallel changes in the N␤25 level. These results demonstrated that the characteristics of C-terminal elongation of N␤ and A␤ are almost identical. In addition, because many other type 1 membrane-bound receptors release intracellular domains by PS-dependent intramembrane proteolysis, we suspect that the release of A␤-or N␤-like peptides is a common feature of the proteolysis during RIP signaling. We anticipate that this study will open the door to searches for markers of RIP signaling and surrogate markers for A␤42 production.

Evidence that Enzyme Processivity Mediates Differential Aβ Production by PS1 and PS2

“…First, they indicate that PS1 and PS2 have similar cleavage activities at the S3/ε-sites of at least two unique substrates, perhaps suggesting a common mechanism by which γ-secretase processes all of its substrates. Furthermore, Notch is cleaved by γ-secretase in a processive manner similar to that of APP [36], so it will be important for future work to determine whether PS1 generates more Notch P3 (also referred to as Notch-β [44, 45]) product than PS2 by successive cleavages culminating at the Notch S4-site, (the corollary to the APP γ-site), and whether Aph1 isoform heterogeneity contributes to processivity on Notch substrate. …”

Amyloid beta 42 peptide (Aβ42)-lowering compounds directly bind to Aβ and interfere with amyloid precursor protein (APP) transmembrane dimerization

“…Indeed, sulindac sulfide and indomethacin decreased the relative level of released Notch-1 Aβ-like peptide species, i.e., Nβ25, without changing the total Nβ level (26). Thus, we assume that binding to the substrate is determined by conformational characteristics like an α-helical flat protein surface, rather than by a specific primary sequence.…”

Substrate Sequence Influences γ-Secretase Modulator Activity, Role of the Transmembrane Domain of the Amyloid Precursor Protein

“…A study by Okochi et al found that ␥-cleavage of a Notch-1-based truncated construct was shifted by treatments with NSAIDs in a manner analogous to that seen in APP by bicineurea gels (45). They reported that sulindac sulfide decreased generation of an N␤ (cognate Notch A␤) fragment terminating in -AAAAFV, whereas we observed that cleavage at this same site in the APP-Notch chimeric construct (1-40) was unchanged by either sulindac sulfide or flurbiprofen.…”