Presenilins mediate a dual intramembranous gamma-secretase cleavage of Notch-1

Abstract

Following ectodomain shedding, Notch-1 undergoes presenilin (PS)-dependent constitutive intramembranous endoproteolysis at site-3. This cleavage is similar to the PS-dependent gamma-secretase cleavage of the beta-amyloid precursor protein (betaAPP). However, topological differences in cleavage resulting in amyloid beta-peptide (Abeta) or the Notch-1 intracellular domain (NICD) indicated independent mechanisms of proteolytic cleavage. We now demonstrate the secretion of an N-terminal Notch-1 Abeta-like fragment (Nbeta). Analysis of Nbeta by MALDI-TOF MS revealed that Nbeta is cleaved at a novel site (site-4, S4) near the middle of the transmembrane domain. Like the corresponding cleavage of betaAPP at position 40 and 42 of the Abeta domain, S4 cleavage is PS dependent. The precision of this cleavage is affected by familial Alzheimer's disease-associated PS1 mutations similar to the pathological endoproteolysis of betaAPP. Considering these similarities between intramembranous processing of Notch and betaAPP, we conclude that these proteins are cleaved by a common mechanism utilizing the same protease, i.e. PS/gamma-secretase.

Fig. 1. Detection of a secreted Notch-1 fragment. (A) Schematic representation of NΔE, NICD and F-NEXT. All three mouse Notch-1 variants contain a hexametric myc tag at the C-terminus to facilitate detection. F-NEXT is a NΔE variant that contains an insertion of the FLAG epitope and two adjacent methionine residues after the signal peptide sequence to facilitate the detection of secreted Notch-1 fragments. An arrowhead indicates the M1727V mutation present in NΔE (Kopan et al., 1996). An arrow shows S3, and the critical valine is shown in bold. The recognition site of the anti-FLAG antibody M2 is represented by the black bar. (B) Detection of a secreted FLAG-tagged Notch-1 fragment (F-Nβ) derived from F-NEXT. Untransfected K293 cells or K293 cells stably expressing NΔE or F-NEXT were pulse labeled with [³⁵S]methionine for 1 h and chased for 0 and 2 h. Upper panel: cell lysates were immunoprecipitated with anti-myc antibody 9E10. F-NEXT and NΔE undergo S3 cleavage with similar efficiency. Note that in this pulse–chase paradigm, NICD generation is detectable after pulse labeling for 1 h without chase. Lower panel: conditioned media from K293 cells stably expressing NΔE or F-NEXT were immunoprecipitated with anti-FLAG M2 agarose. (C) Time-dependent secretion of F-Nβ. K293 cells stably expressing F-NEXT were pulse labeled with [³⁵S]methionine for 1 h and chased for the indicated times. F-Nβ was analyzed in conditioned media, and cell lysates by immunoprecipitation with anti-FLAG M2 agarose. A longer exposure revealed that very low amounts of F-Nβ were also detectable in the cell lysates (data not shown). Identical results were obtained with F-NEXT M1727V (data not shown).

Fig. 2. Characterization of F-Nβ by IP/MS. (A) MALDI-TOF MS spectrum of F-Nβ. Conditioned medium from K293 cells stably expressing F-NEXT was immunoprecipitated with anti-FLAG M2 agarose, and F-Nβ was analyzed by MALDI-TOF MS (see Materials and methods). Molecular masses in Daltons of the individual peaks are indicated. Multiple peaks including a major peak at 3832 Da were observed at the mass range from 3400 to 4400 Da (inset). Peaks higher than 4400 Da including the theoretically predicted peak of ∼5058 Da corresponding to F-Nβ derived from S3 cleavage (asterisk, see B) were not observed. The major F-Nβ peak of 3832 Da was also identified by IP/MS analysis from conditioned media of K293 cells stably transfected with F-NEXT M1727V (data not shown). (B) Schematic representation of intramembranous cleavages of the mouse Notch-1 and human βAPP TMs. S4 cleavage of F-NEXT derivatives occurs predominantly between Ala1731 and Ala1732.

Fig. 3. F-Nβ is generated in a PS- and γ-secretase-dependent manner. (A) PS dependence of F-Nβ generation. K293 cells stably expressing wild-type PS1 or mutant PS1 D385N were stably transfected with the F-NEXT cDNA. Upper panel: F-Nβ generation was analyzed from conditioned media of metabolically labeled cells as described in Figure 1B. Lower panel: corresponding cell lysates were analyzed for NICD generation as described in Figure 1B. (B) γ-secretase dependence of F-Nβ generation. Upper panel: F-Nβ generation was analyzed as described in Figure 1B from conditioned media of K293 cells stably co-expressing F-NEXT and wild-type PS1 that were metabolically labeled in the presence or absence of γ-secretase inhibitor L-685,458 (1 µM). Lower panel: corresponding cell lysates were analyzed for NICD generation as described in Figure 1B.

Fig. 4. PS1 FAD mutations affect the relative levels of elongated F-Nβ species. (A) MALDI-TOF MS spectra of F-Nβ species secreted from cells co-expressing F-NEXT and the indicated PS1 derivatives. Conditioned media were analyzed by IP/MS as described in Figure 2B. The mass range from ∼3750 to ∼4250 Da is shown. Black arrowhead, F-Nβ1731; colored arrowheads, F-Nβ1733 (green), F-Nβ1734 (blue) and F-Nβ1735 (red). The rhombus indicates the peak corresponding to F-Nβ1732 (molecular mass 3903 Da), and the asterisk indicates an F-Nβ1731 (molecular mass 4045 Da) species with a different N-terminus from that of the major F-Nβ1731 (molecular mass 3832 Da) species (compare Figure 2B and Table I). Note that FAD-associated PS1 mutations, in particular PS1 L166P, show an altered production of the various F-Nβ species. (B) C-termini of F-Nβ species affected by FAD-associated PS1 mutants. Sequences within the putative TM are shown. Black, F-Nβ1731; green, F-Nβ1733; blue, F-Nβ1734; red, F-Nβ1735. Arrows indicate S3 and S4. (C) Semi-quantitative analysis of elongated F-Nβ species. Conditioned media from cells expressing the indicated PS1 FAD-associated mutants (each medium amount for immunoprecipitation was normalized to contain the same level of F-Nβ1731; see Materials and methods) were analyzed by IP/MS. Peak heights corresponding to secreted F-Nβ1733, F-Nβ1734 and F-Nβ1735 were measured and are expressed relative to the peak height of the internal control (1 pmol bovine insulin β-chain; see Materials and methods). Asterisks indicate the significance of the increase and decrease of the various F-Nβ species relative to both endogenous/wild-type PS1-expressing cells (P < 0.0001; Students t-test). The data are the mean from five independent measurements.

Fig. 5. Similar intramembranous cleavages of Notch-1 and βAPP. Following S2 cleavage, the Notch fragment NEXT is cleaved in a PS- and γ-secretase-dependent manner within the membrane at two major sites, S3 and S4. S3 cleavage occurs close to the cytosolic membrane border and leads to the liberation of NICD, whereas S4 cleavage occurs near the middle of the TM and causes the release of Nβ peptides. S3 and S4 cleavages are strikingly similar to γ₄₉ and γ₄₀ cleavages of CTFβ of βAPP, respectively. The generation of longer forms of Nβ and Aβ peptides (Aβ_42,43) are affected in a similar manner at least by some FAD-associated PS mutants.