From the Department of Neurobiology, Pharmacology and
Physiology, The University of Chicago, Chicago, Illinois 60637 and the
¶ Department of Human Genetics, The Mount Sinai School of
Medicine, New York, New York 10029
Received for publication, September 10, 2002, and in revised form, November 19, 2002
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ABSTRACT |
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Presenilin 1 (PS1) plays an essential role in
intramembranous " Presenilin 1 and 2 (PS1 and
PS2)1 are polytopic membrane
proteins that are mutated in the majority of pedigrees with early onset
familial Alzheimer's disease (FAD) (1-3). It is now established that
PS play an essential role in intramembranous " Although the mechanism(s) by which PS facilitates While appealing, the "PS is Intrigued by the apparent discordance between the activities of
FAD-linked PS1 mutants on the production of A Cell Culture and Transfection--
Mouse neuroblastoma N2a cells
that constitutively expresses human Swedish APP695 (N2a swe.10) (35)
were maintained in 50% Dulbecco's modified Eagle's medium and 50%
Opti-MEM (Invitrogen) supplemented with 5% fetal bovine serum. To
generate stable cell lines expressing wild-type PS1, the FAD-linked
Conditioned media were collected and immediately adjusted to 0.5 mM phenylmethylsulfonyl fluoride. Cultured cells were lysed in 1× immunoprecipitation (IP) buffer containing 50 mM
Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM EDTA,
0.5% Nonidet P-40, 0.5% sodium deoxycholate, and protease inhibitor
mixture (Sigma). Nuclei and debris were removed by centrifugation and
the protein concentration of detergent-soluble proteins in each lysate
was determined using a bicinchoninic acid protein assay kit (Pierce).
Immunoprecipitation, Electrophoresis, and
Immunoblotting--
For immunoprecipitations, we used equivalent
volumes of conditioned medium based on the calculation of the protein
concentration in each plate of cells to avoid experimental bias because
of variations in cell density. Normalized conditioned media were
immunoprecipitated with 26D6, a monoclonal antibody raised against
A
Aliquots of detergent lysate were fractionated on high percentage
Tris-Tricine SDS-PAGE gels for detection of full-length APP and
APP-CTFs, or Tris glycine SDS-PAGE for analysis of PS1. To detect
secreted A Metabolic Labeling and Immunoprecipitation--
N2a cells were
starved for 30 min in methionine-free Dulbecco's modified Eagle's
medium (Invitrogen) and then labeled with 250 µCi/ml
[35S]methionine (PerkinElmer Life Sciences) in
methionine-free Dulbecco's modified Eagle's medium supplemented with
1% dialyzed fetal bovine serum (Invitrogen) for 10 min (for
pulse-labeling) or 2 h. Conditioned medium was collected and cells
were lysed in IP buffer. For immunoprecipitations we used a volume of
conditioned medium that was normalized to the calculated
trichloroacetic acid-precipitable radioactive counts (cpm) in cell
lysates. Soluble APP Mass Spectrometric Analysis--
Conditioned media
from N2a swe.10 cell pools stably expressing wild-type PS1 or the
PS1 Effects of FAD-linked PS1 Variants on Production of A
Our finding that two FAD-linked PS1 variants enhance secretion of the
principal A Dose Dependence of
Densitometric quantification of the data in Fig. 2A reveals
that the production of A
In parallel to the examination of secreted A MALDI-TOF Analysis of A
It should be noted that while the IP-MS paradigm is useful for the
identification of A Taken together with the finding that intramembranous In an attempt to clarify the effects of FAD-linked mutant PS1 on
the generation of A In this report, we offer several novel insights relevant to
PS-dependent Second, we provide unequivocal evidence that the production of
A Finally, it is now firmly established that PS1 is present in a high
molecular weight complex, and that molecules, termed nicastrin (25),
APH-1-secretase" processing of several type I
membrane proteins, including the
-amyloid precursor proteins (APP)
and Notch1. In this report, we examine the activity of two familial
Alzheimer's disease-linked PS1 variants on the production of
secreted A
peptides and the effects of L-685,458, a potent
-secretase inhibitor, on inhibition of A
peptides from
cells expressing these PS1 variants. We now report that PS1 variants
enhance the production and secretion of both A
1-42 and A
1-40
peptides. More surprisingly, whereas the IC50 for
inhibition of A
1-40 peptide production from cells expressing
wild-type PS1 is ~1.5 µM, cells expressing the PS1
E9 mutant PS1 exhibit an IC50 of ~4 µM.
Immunoprecipitation and matrix-assisted laser desorption/ionization
time-of-flight mass spectrometry reveal that the levels of A
1-43
peptides are elevated in medium of PS1
E9 cells treated with higher
concentrations of inhibitor. The differential effects of wild-type and
mutant PS1 on
-secretase production of A
peptides and the
disparity in sensitivity of these peptides to a potent
-secretase
suggest that PS may be necessary, but not sufficient, to catalyze
hydrolysis at the scissile bonds that generate the termini of A
1-40
and A
1-42 peptides.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-secretase" processing of type I membrane proteins, including the
-amyloid precursor proteins (APP) (4, 5), the developmental signaling receptor,
Notch1 (6-8), the tyrosine kinase receptor ErbB4 (9, 10), and N- and
E-cadherins (11). For APP,
-secretase catalyzes proteolysis of a set
of membrane-tethered APP derivatives, termed APP-CTFs, resulting in the
production and secretion of
-amyloid (A
) peptides. On the other
hand,
-secretase-mediated processing of the membrane-tethered Notch1
derivative, termed S2/NEXT, releases the intracellular domain (S3/NICD)
that subsequently translocates to the nucleus and activates
transcription of target genes (7, 8). The observation that A
and
S3/NICD production are completely eliminated in cells derived from
mouse blastocysts with compound deletions of PS1 and
PS2, lends convincing support to the notion that PS are
critical for intramembranous cleavage of APP and Notch1 (12, 13).
-secretase
processing of APP and Notch1 have not been fully elucidated, the
generation of A
(14-16) and S3/NICD (17) have been show to be
inhibited by highly potent and selective aspartyl protease transition
state inhibitors that bind specifically to PS1 and PS2 (14, 18). These
data, taken with the description of a family of signal peptide
peptidases with limited homology to PS (19), have led to the
conclusion that PS are the elusive
-secretases (20).
-secretase" model has several
weaknesses. First, mutagenesis studies have revealed that
-secretase has relaxed substrate selectivity within the APP transmembrane domain
and occurs at heterogeneous sites (21, 22), while
-secretase cleavage of Notch1 is highly sequence-specific and appears to generate
a single S3/NICD species (7). Second, whereas endocytosis and recycling
of APP-CTFs are required for the generation of A
(23), S3/NICD
production does not require endocytic trafficking of the Notch
derivative, S2/NEXT (24). Third, the identification of several
PS-interacting membrane proteins, including nicastrin (25), APH-1 (26),
and PEN2 (27) that also modulate production of S3/NICD (25-27) and
A
(25, 28) suggests that a protein complex, comprised of PS and
other factors are required for intramembranous proteolysis of APP and
Notch1. Finally, PS1 harboring a substitution of aspartate
257 with alanine is capable of processing APP to A
peptides (29,
30), but fails to generate S3/NICD from a truncated Notch1 molecule,
termed Notch
E (31). Similarly, expression of several FAD-linked
PS1 variants (30-32) or the experimental L286E or L286R
PS1 mutants (33) leads to exaggerated overproduction of
highly fibrillogenic A
42 peptides, but surprisingly, these PS1 variants fail to generate S3/NICD from Notch
E.
42 peptides and S3/NICD
production, we examined the activity of these FAD-linked PS1 variants
on the production of secreted A
peptides and the effects of a potent
aspartyl protease transition state inhibitor of
-secretase, termed
L-685,458 (15, 16) on the production of these A
species. We now
report that while PS1 variants enhance production of A
42, as
expected, there is an unexpected enhancement in levels of secreted
A
40 peptides. We also provide the first demonstration that in the
conditioned medium of "pools" of stable cell lines that express
individual FAD-linked mutant PS1, both A
1-40 and 1-42 peptides
accumulate to higher levels than the A
peptide variants in medium of
cell pools that express wild-type PS1. More surprisingly, under
conditions at which the
-secretase inhibitor completely eliminates
production of all A
-related peptides from cells expressing wild-type
PS1, we now report that the inhibitor is not fully effective at
lowering production of A
variants from cells expressing two
independent FAD-linked PS1 mutants. Hence, we argue that production of
A
peptides are differentially regulated by the expression of
wild-type and FAD-linked PS1 variants.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
E9, or E280A variants, N2a swe.10 cells were cotransfected with 10 µg of PS1 cDNAs (in pAG3Zeo vector) and 100 ng of pIREShygro
using the calcium phosphate method (36). Cells expressing transgene
were selected with 400 µg/ml hygromycin. Hygromycin-resistant
colonies were further screened in medium containing 400 µg/ml zeocin
(Invitrogen) to generate a stable pool. Approximately 100-200
zeocin-resistant colonies were pooled and analyzed.
-Secretase Inhibitor Assay--
For
-secretase inhibitor
assays, cells were incubated for 16 h in medium containing 2 µM (or indicated concentrations) of the
-secretase
inhibitor, L-685,458 (16), prepared in dimethyl sulfoxide
(Me2SO) or an equivalent concentration of Me2SO
as a vehicle control.
1-12 (37), for 16 h at 4 °C. The immune complexes were
"bridged" by the addition of rabbit anti-mouse IgG (Pierce),
collected with protein A-conjugated agarose beads (Pierce), and eluted
by boiling for 5 min in Laemmli SDS sample buffer prior to
fractionation on SDS-PAGE.
40/42, immunoprecipitated samples were fractionated on
Bicine/urea gels (38). Fractionated proteins were electrophoretically transferred to polyvinylidene difluoride membranes (Bio-Rad), and the
membranes were probed with appropriate primary antibodies. Full-length
APP and APP-CTF were detected by CT15, an antisera that recognizes the
carboxyl-terminal 15 amino acids of APP (39). A polyclonal antibody,
PS1NT, was used to detect full-length PS1 and PS1 NTF (40).
Soluble APP
and A
40/42 were detected by 26D6 (37). After
incubation with horseradish peroxidase-coupled secondary antibodies
(Pierce), bound antibodies were visualized using an enhanced
chemiluminescence (ECL) detection system (Perkin-Elmer Life Sciences).
and A
40/42 were immunoprecipitated with
monoclonal antibody, 26D6 (37). To examine APP synthesis, cells were
pulse-labeled with [35S]methionine for 10 min, and APP
was immunoprecipitated with 369 antibody, raised against a peptide
corresponding amino acids 649-695 of APP695 (41).
Immunoprecipitates were fractionated by SDS-PAGE, and radioactive bands
were visualized and quantified using a PhosphorImager (Amersham Biosciences).
E9 were immunoprecipitated with 4G8 antibody, specific for amino
acids 17-24 of A
, and collected with Protein A/G-coupled
agarose beads prior to analysis by matrix-assisted laser
desorption/ionization time-of-flight (MALDI-TOF) mass spectrometric analysis, as described (42). Each mass spectrum was averaged from at
least 500 measurements, and bovine insulin was included as an internal
mass calibrant.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Variants
and Inhibition with a
-Secretase Inhibitor--
It is now well
accepted that expression of FAD-linked PS1 variants elevate the levels
of secreted A
1-42 peptides and, in so doing, increase the
calculated ratio of A
40/A
42 peptides. However, the absolute
levels of A
peptide variants have rarely been reported, a
reflection, in large part, of the variability in transgene-encoded APP
between individual lines. With this caveat, we chose to transfect a
neuroblastoma N2a cell line that constitutively expresses a
carboxyl-terminal Myc epitope-tagged human APP 695 harboring the
FAD-linked "Swedish" mutations (38) with human wild-type PS1
(wtPS1) or the FAD-linked PS1 variants, PS1
E9, or E280A to generate
stable pools that express human PS1 polypeptides. Western blot analysis
of stable cell pools revealed the accumulation of human PS1 NTF and low
levels of full-length precursor in cells expressing wild-type PS1 (Fig.
1A, lane 1),
uncleaved ~43-kDa PS1
E9, and low levels of endogenous mouse PS1
NTF in cells expressing PS1
E9 (Fig. 1A, lane
2), and mutant human PS1 NTF and low levels of full-length
precursor in cells expressing the E280A variant (Fig. 1A,
lane 3). In these cell pools, "replacement" of the bulk of murine PS1 fragments has occurred (45), although residual levels of
murine NTF (as seen in the PS1
E9 cells) are still present. This
would be expected in a cell pool in which transgene-derived products
are expressed at varying levels in independent clones. We examined the
levels of secreted A
-related species by immunoprecipitation with
antibody 26D6 (39), specific for A
residues 1-12, fractionation of
immune complexes on Bicine/urea gels, and analysis of
immunoprecipitated A
peptides using 26D6 antibody and enhanced
chemiluminescence detection. For these studies, we calculated the
protein concentration in each plate of cells, and used normalized
volumes of medium so that there would be no experimental bias because
of differences in cell density. In Fig. 1, we show that constitutive
expression of wild-type PS1 leads to robust secretion of A
1-40
peptides, limited levels of secreted A
1-37, A
1-38,
and A
1-39 peptides and nearly undetectable levels of A
1-42
peptides (Fig. 1B, lane 1). This level of
secreted A
peptides is no higher than parental APPswe.10 cells (data
not shown). On the other hand, we consistently observed that the levels
of accumulated A
1-40 and A
1-42 peptides were elevated in medium
of cells expressing either the PS1
E9 or A280E variants (Fig.
1B, lanes 3 and 5, respectively). Even more surprising was the observation that under conditions in which treatment of wtPS1 cells with a potent
-secretase inhibitor, L-685,458 (17) (2 µM for 16 h), resulted in nearly
complete inhibition of secreted A
peptides (Fig. 1B,
lane 2), low levels of A
1-40 and A
1-42 peptides
still remained in the medium of cells expressing either PS1 mutant
(Fig. 1B, lanes 4 and 6).
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Fig. 1.
Effects of FAD-linked PS1 variants on
production of secreted A peptides.
A, expression of PS1 in N2a swe.10 pooled cells that
stably express wild-type PS1 (lane 1), FAD-linked PS1
E9
(lane 2), or E280A (lane 3) were analyzed by
immunoblotting with PS1NT antiserum. B, N2a
swe.10 pooled cells that express wild-type PS1 (lanes 1 and
2), FAD-linked PS1
E9 (lanes 3 and
4), or E280A (lanes 4 and 5) were
incubated for 16 h in the absence (lanes 1,
3, and 5) or presence (lanes 2,
4, and 6) of
-secretase inhibitor,
L-685,458 (2 µM). Conditioned medium was
immunoprecipitated with 26D6 and immune complexes were subjected to
electrophoresis in Bicine/urea gels, followed by immunoblotting with
26D6; A
1-42 exhibits more rapid migration than A
1-40 in this
gel system (39). Note that secreted levels of both A
1-40/1-42
peptides appears elevated in cells expressing either the PS1
E9
(lane 3) or A280E (lane 5) variants. Treatment
with the
-secretase inhibitor resulted in marked inhibition of
secreted A
peptide production in cells expressing wild-type PS1
(lane 2), whereas there was a considerable level of A
peptides, most notably A
1-42 species, still remaining in medium of
cells expressing the PS1
E9 (lane 4) or E280A (lane
6) variants. C, APP synthesis was examined by
10-min pulse labeling with [35S]methionine followed by
immunoprecipitation using 369 and phosphorimaging. Note that APP
synthesis is indistinguishable between the pooled cells expressing
either wild-type PS1 (lane 1) or PS1
E9 (lane
2). D, N2a swe.10 pooled cells expressing
wild-type PS1 (lanes 1 and 2) and the PS1
E9
(lanes 3 and 4) were metabolically labeled with
[35S]methionine for 2 h in the absence (lanes
1 and 3) or presence (lanes 2 and
4) of the
-secretase inhibitor. Conditioned medium was
immunoprecipitated with 26D6 and separated by Tris-Tricine SDS-PAGE.
APPs
generated by
-secretase and A
1-40/A
1-42
peptides are indicated. Note that the level of total secreted A
peptides in medium of cells expressing the PS1
E9 are elevated
relative to the A
peptides in wild-type PS1 cells.
E, to examine the identity of secreted A
species,
A
radiolabeled from cells expressing wild-type PS1
(lanes 1 and 2) or the FAD-linked PS1
E9
(lanes 3 and 4) was subjected to
immunoprecipitation with 26D6, and immune complexes were fractionated
on Bicine/urea gels. Note that dramatic effects of the inhibitor are on
the A
1-40 species, with relative sparing of A
1-42 species
(lane 4). F, the absolute PhosphorImager
units of the bands corresponding to A
1-40 (left panel)
and A
1-42 (middle panel) were determined by
phosphorimaging, and the ratio of counts/min in A
42 to A
40 was
plotted (right panel). The asterisk in the
middle and right panels reflects the undetectable
levels of A
42 in medium of wtPS1 cells in the presence of the
inhibitor.
variant, A
1-40, is novel and we felt it important to
fully validate this finding. We chose to focus on the PS1
E9 pool.
First, to establish that the differences in accumulated A
peptides
between wtPS1 and PS1
E9 cell pools was not simply a reflection of
differences in synthetic levels of transgene-encoded APPswe, we
pulse-labeled cells with [35S]methionine for 10 min and
analyzed newly synthesized APP in cell lysates by subjecting equivalent
detergent-soluble, trichloroacetic acid-precipitable, radioactivity
(cpm) to immunoprecipitation with antibody 369, raised against a
peptide corresponding to amino acids 649-695 of APP (43),
fractionation of immune complexes on SDS-PAGE, and phosphorimaging.
In Fig. 1C, we show that the synthesis of full-length APP is
indistinguishable between the cell pools that express either human
wtPS1 or PS1
E9. To further quantify the absolute increase in both
A
1-40 and A
1-42 peptides in medium of mutant PS1-expressing
cells relative to cells expressing wtPS1, we incubated cell pools with
[35S]methionine for 2 h and quantified the levels of
secreted A
peptides in medium by immunoprecipitation with antibody
26D6, fractionation of immune complexes on Tris-Tricine gels, and
phosphorimaging. For these analyses, we quantified total counts/min in
detergent-solubilized cell lysates and used normalized volumes of
radiolabeled conditioned medium for immunoprecipitations. As we have
shown by Western blot analysis (Fig. 1B), quantitative
phosphorimaging analysis revealed an elevation in total A
peptides
in medium of cells expressing the PS1
E9 mutant (Fig. 1D,
lane 3) by 2.8-fold relative to A
peptides secreted from
cells expressing wtPS1 (Fig. 1D, lane 1). Notably, the 26D6 antibody, specific for A
residues 1-12, also detects soluble derivatives generated by
-secretase, termed
APPsa, quantitative phosphorimaging revealed a 1.5-fold
increase in levels of APPsa in medium of PS1
E9 cells
relative to cells expressing wild-type PS1 (Fig. 1D, compare
lanes 3 and 1, respectively), this despite identical synthetic rates of the APPswe precursor between cell pools
(Fig. 1C). These findings offer the suggestion that
expression of the PS1
E9 variant leads to enhanced trafficking (or
processing) of full-length APPswe to cellular compartments in which
-secretase is active, but further studies will be
necessary to address this issue. In any event, quantitative
phosphorimaging revealed that while treatment of wtPS1 cells with 2 µM inhibitor reduced production of newly synthesized A
peptides to ~0.4% of untreated controls (Fig. 1D,
lane 2), the inhibitor diminished the levels of total A
peptide species in medium of PS1
E9 cells to ~12% of the untreated control (Fig. 1D, lane 4). Further examination of
the complexity of radiolabeled A
peptide variants by Bicine/urea
gels (Fig. 1E) revealed that the absolute levels of both
A
40 and A
42 variants were elevated in medium of PS1
E9 cell
medium (Fig. 1E, lane 3; quantified in Fig.
1F, left panel) relative to the levels in medium of cells expressing wild-type PS1 (Fig. 1E, lane
1; quantified in Fig. 1F, left panel).
Furthermore, under conditions where the
-secretase inhibitor almost
completely eliminated A
1-40 species in medium of cells expressing
wild-type PS1, this compound diminished A
1-40 peptide levels to
~13% in medium of cells expressing PS1
E9 (Fig. 1E;
quantified in Fig. 1F, left panel). Similarly,
the
-secretase inhibitor fully eliminates secreted A
1-42
peptides from cells expressing wild-type PS1, but L-685,458 treatment
only reduced the level of A
1-42 peptides to ~58% in medium of
cells that express PS1
E9 (Fig. 1E, lane 4;
quantified in Fig. 1F, middle panel). Notably,
the levels of A
1-40 and A
1-42 peptides are nearly identical in
PS1
E9 cell medium after treatment with L-685,458 (Fig.
1F, right panel).
-Secretase Inhibitor on Secretion of A
Peptide Variants--
Having established that treatment with 2 µM L-685,458 for 16 h completely eliminates A
peptides production from cells expressing wtPS1 cells, but fails to
fully inhibit production of secreted A
peptide from cells expressing
PS1
E9 cells, we asked whether the individual A
peptide variants
may be differentially sensitive to the dose of
-secretase inhibitor.
We treated parallel dishes of N2a swe.10 cell pools expressing
wild-type PS1 or the PS1
E9 variant with increasing concentrations of
L-685,458 for 16 h. The conditioned medium was collected and the
protein concentration in cell lysates was determined. Normalized
volumes of medium, relative to protein concentration in
cell lysates, were subjected to immunoprecipitation and Western blot
analysis. Consistent with the data shown in Fig. 1, the constitutive
levels of A
1-40 and A
1-42 peptides in medium of cells
expressing the PS1
E9 variant are elevated compared with cells
expressing wild-type PS1 (Fig. 2A, compare lanes 6 and 1, respectively). At 2 µM, L-685,458
lowered the levels of secreted A
1-40 peptides in medium of wtPS1
cells to ~20% of the levels in vehicle-treated cells (Fig.
2A, lane 3). However, at this concentration of
inhibitor, the levels of A
1-40 peptides in medium of cells
expressing PS1
E9 were reduced to ~75% of the levels in
vehicle-treated cells (Fig. 2A, lane 8). Notably,
treatment with 8 µM inhibitor virtually eliminates all
A
peptides in medium of cells expressing wild-type PS1 (Fig. 2A, lane 5), but this concentration of inhibitor
diminished the levels of A
1-40 peptides in medium of PS1
E9 cells
to ~20% of the level in vehicle-treated cells (Fig. 2A,
lane 10).
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Fig. 2.
Effects of FAD-linked
E9 on inhibition by a
-secretase inhibitor. A, N2a swe.10
cells stably expressing wild-type PS1 (lanes 1-5) or the
PS1
E9 (lanes 6-10) were incubated for 16 h in
serum-free medium containing the indicated concentrations of
-secretase inhibitor, L-685,458. Note that production of secreted
A
in cells expressing wild-type PS1 was largely inhibited by
treatment between 2 and 4 µM (lanes 3 and
4), whereas a significant amount of A
peptides was
detected at these concentrations of the inhibitor in cells expressing
PS1
E9 (lanes 8 and 9). Notably, treatment of
the inhibitor at 8 µM fails to diminish the level of A
peptides in medium of the PS1
E9 cells (lane 10), whereas
treatment at this concentration of the inhibitor virtually eliminates
A
peptides in wild-type PS1 cells (lane 5).
B, the band intensity corresponding to A
1-40
(open circle) and A
1-42 (closed circle) were
quantified using densitometry (Molecular Dynamics) and plotted.
C, accumulation of APP-CTFs in detergent lysates
prepared from cells expressing wild-type PS1 (lanes 1-5) or
the PS1
E9 (lanes 6-10) were analyzed by immunoblotting
with CT15 antibody. Note that both endogenous APP-CTF
and
APPswe-derived
- and
-CTFs accumulate even at 1 µM
inhibitor in cells expressing wild-type PS1 (lane 2), and
that the level of these species does not change as the concentration of
the inhibitor is increased (lanes 3-5). Treatment of the
inhibitor at 1 µM resulted in lower levels of accumulated
APP-CTFs (lane 7) in cells expressing the PS1
E9 compared
with those observed at the same concentration in wild-type
PS1-expressing cells.
1-40 and A
1-42 in cells expressing
wild-type PS1 are equally sensitive to the
-secretase inhibitor,
with an IC50 of ~1.5 µM (Fig.
2B, left panel). On the other hand, the production of A
1-40 is inhibited with an IC50 of over 4 µM in cells expressing PS1
E9 (Fig. 2B,
right panel). Moreover, in medium of PS1
E9
cells, 50% inhibition of A
1-42 peptides occurs with 2 µM inhibitor (also apparent by immunoprecipitation of
radiolabeled A
1-42 peptides; Fig. 1, E and
F), but at higher concentrations of compound, there is a
paradoxical plateau in levels of these peptides. However, MALDI-TOF
analysis (see below, Fig. 3) of the peptides generated after treatment of PS1
E9 cells with high
concentrations of compound reveal that A
1-43 peptides now become
prominent. We do not know where A
1-43 peptides migrate in
Bicine/urea gels, but it is likely that these overlap with the
A
1-42 species and hence, we argue that the Western blot signals in
lanes 9 and 10 of Fig. 2A represent a
combination of A
1-42 and A
1-43 variants.
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Fig. 3.
Mass spectrometric analysis of secreted
A . N2a swe.10 pooled cells stably expressing
wild-type PS1 (panels A-D) or the PS1
E9 (panels
E-H) were incubated for 16 h in serum-free medium containing
Me2SO alone (panels A and E), 1 µM (panels B and F), 4 µM (panels C and G), or 8 µM (panels D and H) L-685,458. The
experimental mass and peptide lengths for several A
peptide variants
are shown in panels A and E. Peaks that cannot be
identified as A
-related are designated as "n."
peptides, we examined
the accumulation of APP-CTFs in detergent-solubilized lysates from
cells treated with increasing concentrations of the
-inhibitor. In
cells that express wild-type PS1, both endogenous
-CTF, and
APPswe-derived
- and
-CTFs accumulate even after treatment with 1 µM inhibitor (Fig. 2C, lane 2), and
the level of these fragments seems not to increase as a function of
inhibitor concentration (Fig. 2C, lanes 3-5). In
contrast, treatment of PS1
E9 cells with 1 µM inhibitor
leads to the accumulation of APP-CTFs (Fig. 2C, lane
7), but at considerably lower levels than that observed in lysates
of wtPS1 cells treated with the same concentration of inhibitor.
Indeed, the most robust increase in accumulated APP-CTFs in
PS1
E9-expressing cells occurs after treatment with inhibitor at 4 and 8 µM (Fig. 2C, lanes 9 and
10, respectively), concentrations that also appear to have
the most pronounced effect on A
production (Fig. 2A,
lanes 9 and 10).
Variants in Medium of Cells Expressing
Human Wild-type PS1 or PS1
E9 following Treatment with a
-Secretase Inhibitor--
To characterize the A
-related species
that accumulate in medium of cells expressing human wild-type PS1 and
PS1
E9 cells, and in medium of these cell pools treated with
differing concentrations of the inhibitor, we used antibody 4G8,
specific for epitopes 17-24 of A
, to immunoprecipitate A
-related
peptides from several of the samples shown in Fig. 2; resulting
immune complexes were analyzed by MALDI-TOF mass spectrometry (42). In
Fig. 3, panel A, we show that in medium of wild-type PS1
cells treated with vehicle (Me2SO), A
1-40
(Mr 4330) is the prominent species, with minor
species of 1-34, 1-37, 1-38, and 1-39 also present. Upon treatment
of these cells with 1 µM L-685,458, A
1-40 and the
minor species are still present, and a small peak, representing
A
1-42 (Mr 4514) also appears (Fig. 3,
panel B); low concentrations of
-secretase inhibitors
have been shown to have a paradoxical effect on elevating A
1-42
peptides (44). However, the levels of all peptides are dramatically
reduced after treatment at 4 µM (Fig. 3, panel
C), and virtually nonexistent after treatment with 8 µM L-685,458 (Fig. 3, panel D). In medium of
untreated PS1
E9 cells, we observed the presence of both A
1-40
and A
1-42 peptides and trace levels of A
1-38, A
1-39, and
A
1-43 peptides (Fig. 3, panel E). After treatment with 1 µM L-685,458, we failed to see an appreciable difference
in the levels of any of the aforementioned A
species (Fig. 3,
panel F). However, and in sharp contrast to our observations
of A
peptides in wtPS1 cell medium, treatment of PS1
E9 cells with
4 µM L-685,458 lead to a reduction in levels of A
40
peptides, with no appreciable change in the levels of A
1-42 and
A
1-43 species (Fig. 3, panel G). Most interestingly, we
now observe a pronounced elevation in levels of A
1-43 peptides. This latter result becomes much more apparent in PS1
E9 cells treated
with 8 µM L-685,458, where the levels of A
1-43
peptide now exceeds the levels of A
1-42 peptides (Fig. 3,
panel H); significant levels of the A
1-40 peptide are
still apparent under these conditions. Furthermore, and in sharp
contrast to cells expressing wild-type PS1 (Fig. 3, panels
B-D), the levels of A
1-34, A
1-37, A
1-38, and A
1-29
variants in medium of PS1
E9 cells appear unchanged no matter what
concentration of inhibitor is employed.
peptides, the technique only serves a
qualitative tool for assessment of peptide levels. For example, the
levels of A
1-40 peptides in lanes 7 and 9 of
Fig. 2 with the analogous samples analyzed by IP-MS in Fig. 3,
panels F and G, respectively, are not
comparable. It is highly likely that under the conditions used in the
IP-MS experiment, the antibody is not in excess, and hence, the
antibodies are fully saturated with captured A
-related peptides.
Moreover, it is our experience, and those of others, that unusual
biophysical properties (including aggregation) of A
1-42 peptides
greatly hinder desorption of these species from the matrix and thus,
the peak heights observed in MALDI-TOF analysis do not accurately
reflect steady-state levels. This artifact is likely exaggerated for
A
1-43 peptides. Hence, the signals observed in Western blot
analysis of fractionated A
peptides on Bicine/urea gels provide a
more accurate reflection of the steady-state levels of A
species in
the medium.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-secretase
processing of APP and Notch1 is abolished in cells with genetic ablations of PS1 and PS2 (12, 13), and the
demonstration that high affinity
-secretase inhibitors specifically
bind to PS (14, 18), it has been concluded that PS are the elusive
-secretases (20). However, several lines of evidence have emerged
that have questioned the veracity of the PS are
-secretase model.
First, intramembranous processing of APP and Notch1 can be
discriminated by a JLK family of nonpeptidic inhibitors (45); the JLK
inhibitors block A
peptide production, but have very little, if any,
effect on production of the Notch1 derivative, S3/NICD (45). Second, expression of the FAD-linked L166P PS1 variant (32) or the
experimental L286E or L286R PS1 mutants (34) leads to
exaggerated overproduction of A
42 peptides, but these PS1
variants fail to generate S3/NICD (32, 34). Supporting these studies,
St. George-Hyslop and colleagues (33) recently reported that expression
of the L392V, G206A, or
E9 variants results in compromised S3/NICD
production and "
" cleavage within the APP transmembrane domain.
Thus, it would appear that while the production of A
peptides,
cleavage at the Notch S3 and APP
sites are
presenilin-dependent, the catalytic activities may be
distinct (33). Finally, recent studies have shown that the generation
of intracellular A
1-42 peptides is PS-independent, suggesting that
intramembranous processing of APP may be mediated by distinct
-secretase activities (46).
peptides, we generated pools of stable cell
lines expressing human PS1 or several independent FAD-linked PS1
variants and analyzed accumulated A
peptide variants in the medium
of these cells. In addition, we assessed the effects of a potent and
highly selective transition state inhibitor of
-secretase on
FAD-linked PS1-mediated production of A
peptides.
-secretase processing of APP. First, we
provide the first demonstration that in the conditioned medium of pools of stable cell lines that express individual FAD-linked mutant PS1,
both A
1-40 and 1-42 peptides accumulate to higher levels than the
A
peptide variants in medium of cell pools that express wild-type
PS1. Despite years of investigation describing the effects of
FAD-linked PS1 on elevation in the relative ratio of A
42:A
40 peptides, it comes as a surprise that the absolute levels of secreted A
species have not been compared in a systematic fashion. In large
part, we suspect that in the analysis of individual stable cell lines,
the levels of coexpressed human APP are highly variable, whether the
APP transgene is coselected, or when the PS1 transgenes are stably
expressed in a "parental" cell line that constitutively expresses a
human APP transgene. In the present study, we utilize cell pools to
"normalize" the level of APP expression across the entire
population of between 100 and 200 individual lines. The mechanism(s)
involved in the exaggerated production of A
1-40 peptides are not
presently known, but may involve PS1
E9 enhancement of cleavage at
the
-secretase site of full-length APPswe. However, using
radiolabeling studies, we have not observed any significant differences
in the production, or accumulation of CTF
in cell pools expressing
either wild-type PS1 or PS1
E9 (data not shown). Thus, we would offer
the tentative conclusion that in addition to enhancing processing at
sites within the APP transmembrane domain to generate A
1-42, the
PS1
E9 variant (and E280A variant) also enhances processing at the
scissile bond between amino acids 636 and 637 (of APP695) to elevate
production of A
1-40 peptides. In support of this suggestion, we
observe that in titration studies using L-685,458 (Fig. 2C),
APP-CTFs only accumulate at higher concentrations of inhibitor in cells
expressing PS1
E9. Thus, it would appear that PS
E9-mediated
intramembranous processing of APP-CTFs is more efficient, thus
enhancing production of A
1-40 and 1-42 peptides, and that these
reactions are less sensitive to concentrations of inhibitor that would
otherwise block A
production in cells expressing wild-type PS1.
1-42 peptides from cells expressing FAD-linked PS1
E9 and E280A
mutations are largely insensitive to a potent
-secretase inhibitor,
L-685,458, at concentrations that would otherwise inhibit A
production from cells that express wild-type PS1. In addition, the
FAD-linked PS1-dependent production of A
1-40 is
somewhat refractory to inhibition by this compound. Similar results
have been obtained using a structurally unrelated
-secretase
inhibitor, compound E (47). Of significant interest is our finding that while the production of A
1-42 peptides are largely resistant to
inhibition by L-685,458, the production of A
1-43 peptides by cells
expressing PS1
E9 are elevated in parallel with increasing concentrations of L-685,458. The molecular mechanisms underlying the
curious observation that A
1-42 peptide production is highly refractory to potent
-secretase inhibitors is perplexing. Earlier studies showed that a peptidomimetic inhibitor, termed compound 1, could block production of both A
40 and 42 peptides with an IC50 of ~16 µM in Chinese hamster ovary
cell lines that stably express human wild-type PS1, and that the
IC50 was slightly increased (to ~22 and ~20
µM for inhibition of total A
and A
42, respectively) in cell lines expressing the FAD-linked M146L PS1 variant (47). The
authors argue that PS1 contains the active site of
-secretase and
that FAD-linked mutations induce subtle changes in PS1 conformation within the proteolytic complex, thus requiring a higher concentration of inhibitor to block A
production. In contrast to these earlier studies, we now show that at the IC50 for inhibition of
A
1-40 production from cells expressing wild-type PS1 (~1.5
µM), L-685,458 only reduces A
1-40 levels by ~25%
from cells expressing PS1
E9. Moreover, the production of A
1-42
peptide production from cells expressing PS1
E9 is highly resistant
to the inhibitor, even at the highest concentrations of compound
tested. At a mechanistic level, a series of recent elegant
enzymological studies of solubilized
-secretase activity that
employed a variety of aspartyl protease transition state analogs
(including L-685,458) and nontransition state analogs have led to the
conclusion that these compounds act in a noncompetitive fashion (48).
The model proposed is that the substrate binding site is distinct from
the active site, but once "docked," the substrate is subsequently
displaced to the catalytic site of the enzyme (48). The model that PS1
harbors the active site of
-secretase, and that FAD mutants subtly
alter PS1 conformation so as to alter the IC50 of
-secretase inhibitors (48) is tempting. However, it remains
extremely perplexing that the FAD-linked PS missense mutations occur
widely throughout the molecule, including in hydrophilic loop domains
that are predicted to be quite remote from the lipid environment in
which intramembranous cleavage takes place. Nevertheless, all of the
FAD-linked PS variants have the unique property of enhancing cleavage
at a single site in the APP transmembrane domain. It is difficult to
reconcile this with a simple effect on an active site. Furthermore,
expression of experimental PS1 variants harboring a P434A mutation in
the PS1 carboxyl-terminal domain (49) or a deletion of the first two
transmembrane domains (50), sequences that are remote from two
transmembrane aspartate residues that are proposed to serve as the
catalytic center (20), also reduces the production of A
peptides,
arguing that multiple domains of PS1 are required for
-secretase
activity (49, 50). Finally, the observation that in cells expressing
FAD-linked mutant PS1, the production of A
1-40 versus
A
1-42 peptides are differentially sensitive to L-685,458 (this
study) makes it difficult to conceive of a model in which hydrolysis at
the scissile bonds that generate the termini of A
1-40 and A
1-42
peptides could be mediated by PS, alone.
/APH-1
(26), and PEN2 (27), are components of this complex.
Moreover, nicastrin and PS levels are coregulated (51-53), and RNA
interference (RNAi)-mediated reduction of nicastrin, APH-1, or
PEN2 levels results in compromised secretion of A
1-40 and A
1-42
peptides (27). Hence, each component of the complex appears to exert
differential effects of A
production. Hence, it is conceivable that
the
-secretase inhibitors block A
production by influencing the
interactions and biochemical properties of individual subunits within
the complex. Future efforts will require the development of
reconstitution systems to elucidate the biochemical interactions of PS
with nicastrin, APH-1, and PEN2 and the effects of these components on
modulation of PS- and FAD-linked PS-mediated
-secretase processing
of APP-CTFs.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Drs. Mark S. Shearman and Yue-Ming Li, Merck Research Laboratories, for providing the aspartylprotease transition state analog, L-685,458.
![]() |
FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grants AG021494 (to S. S. S.) and AG10491 (to R. W.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Recipient of postdoctoral fellowships for research abroad from Japan Society for the Promotion of Science.
To whom correspondence should be addressed: Center for
Molecular Neurobiology, The University of Chicago, Abbott 510, 947 E. 58th St., Chicago, IL 60637. Tel.: 773-834-9186; Fax: 773-834-5311; E-mail: ssisodia@drugs.bsd.uchicago.edu.
Published, JBC Papers in Press, December 19, 2002, DOI 10.1074/jbc.M209252200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
PS, presenilin;
FAD, familial Alzheimer disease;
APP, -amyloid precursor protein;
A
,
-amyloid;
NEXT, Notch extracellular truncation;
NICD, Notch
intracellular domain;
CTF, COOH-terminal fragment;
IP, immunoprecipitation;
NTF, NH2-terminal fragment;
wt, wild-type;
Bicine, N,N-bis(2-hydroxyethyl)glycine;
MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight;
Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.
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