From the Department of Neuropathology and
Neuroscience, the § Department of Synthetic Natural Products
Chemistry, and the ¶ Department of Rational Medicinal Science,
Graduate School of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
Received for publication, February 14, 2003, and in revised form, March 10, 2003
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ABSTRACT |
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Nonsteroidal anti-inflammatory drugs (NSAIDs)
have been known to reduce risk for Alzheimer's disease. In addition to
the anti-inflammatory effects of NSAIDs to block cylooxygenase, it has
been shown recently that a subset of NSAIDs selectively inhibits the
secretion of highly amyloidogenic A Alzheimer's disease
(AD)1 is a dementing
neurodegenerative disorder of the elderly characterized pathologically
by neuronal loss in the cerebral cortex accompanied by massive
deposition of amyloid Mutations in PS1 or PS2 genes account for the
majority of early onset familial AD, and these mutations cause an
increase in the ratio or levels of production of A Epidemiological studies have shown that long term treatment with
nonsteroidal anti-inflammatory drugs (NSAIDs) prevents the development
of AD (reviewed in Refs. 14 and 15). Recently, a prospective,
population-based cohort study provided strong evidence that the long
term use of NSAIDs significantly reduced the risk of AD (16). NSAIDs
affect the inflammatory response by direct inhibition of cyclooxgenase
(COX) enzymes. Moreover, recent studies indicate that NSAIDs are
involved in transcriptional regulation by the modulation of I Compounds and Peptides--
Synthesis of DAPT (23) was performed
by a standard solution phase peptide synthesis utilizing the Cbz
protecting group. The detailed synthetic procedure will be reported elsewhere.
NSAIDs used in this study (SSide, Ssone, and naproxen) were purchased
from Biomol (Plymouth Meeting, PA) and were dissolved in dimethyl
sulfoxide (Me2SO). L-685,458 and standard A Cell Culture and Treatment by NSAIDs--
The expression vectors
encoding Purification of Recombinant Substrates--
cDNAs encoding
the C-terminal 99 amino acids of human Preparation of Solubilized In Vitro Characterization of the
Intramembranous cleavage of Effect of Sulindac Sulfide on
To examine whether SSide modulates
In contrast to the results in cultured cells, the application of SSide
at low concentrations (1-25 µM) caused a transient, but
significant, increase in A
To characterize further the inhibitory mechanism of
We next analyzed the effect of SSide on intracellular domain generation
from recombinant substrates of It has been shown that a subset of NSAIDs selectively lowers the
secretion of A It has been extensively documented that NSAIDs exhibit various
molecular targets, of which the primary target is COX, that converts
arachidonic acid to prostaglandins (17). In addition, SSide has been
shown to inhibit I To date, several The molecular mechanism underlying the reciprocal regulation in
A It has been documented that almost all 42 from cultured cells, although
the molecular target(s) of NSAIDs in reducing the activity of
-secretase for A
42 generation
(
42-secretase) still remain unknown. Here we show that sulindac sulfide (SSide) directly acts on
-secretase and preferentially inhibits the
42-secretase activity derived from the
3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate-solubilized membrane fractions of HeLa cells, in an in vitro
-secretase assay using recombinant amyloid
precursor protein
C100 as a substrate. SSide also inhibits activities for the generation
of A
40 as well as for Notch intracellular domain at higher
concentrations. Notably, SSide displayed linear noncompetitive
inhibition profiles for
42-secretase in
vitro. Our data suggest that SSide is a direct inhibitor of
-secretase that preferentially affects the
42-secretase activity.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
peptides (A
) as senile plaques (1). A
is produced by sequential proteolytic cleavages of the amyloid
precursor protein (
APP) by a set of membrane-bound proteases termed
- and
-secretases. The C-terminal length of A
generated by
-secretase is heterogeneous; A
42 is a relatively minor molecular
species of the A
secreted from cells, but it has a much higher
propensity to aggregate and form amyloid compared with other A
species. These findings provide strong support for the hypothesis that the deposition of A
42 is closely related to the pathogenesis of AD,
implicating
-secretase as an important therapeutic target.
42 (1). It is
known that PS is essential for the
-secretase-mediated
intramembranous cleavage not only for
APP but for other type I
transmembrane proteins (e.g. Notch, ErbB4, E-cadherin,
low density lipoprotein receptor-related protein, and CD44) (2).
PS proteins undergo endoproteolysis to generate N- and C-terminal
fragments and interact with other proteins (i.e. nicastrin,
APH-1, and PEN-2) to form a high molecular weight (HMW) protein complex
(3). The functional role of PS complex in
-secretase activity still
remains unknown. However, aspartyl protease transition state analogue
inhibitors of
-secretase, which harbors a hydroxyl ethylene isostere
or a difluoro alcohol moiety, directly label PS fragments (4-6). In
addition, a systematic analysis using a variety of PS mutants revealed
that HMW complex formation of PS as well as conserved aspartyl residues
within the transmembrane domain are essential for
-secretase
activity (7-11). Finally, in vitro
-secretase activity
is associated with PS HMW complex (12, 13). These data suggest that HMW
PS complex corresponds to the
-secretase, an atypical
membrane-embedded aspartyl protease, and that PS proteins harbor the
catalytic center for
-secretase complex (3).
B
kinase
or peroxisome proliferator-activated receptors (PPAR)
(reviewed in Ref. 17). It has been believed that NSAIDs might influence
the AD pathology by inhibiting the inflammation response
(e.g. activation of microglia and astroglia) in brains (14).
In fact, administration of NSAIDs affects the inflammatory profile and
inhibits the progression of amyloid deposition in the brains of
APP
transgenic mice (18-20). Contrary to these traditional views, however,
it has recently been shown that a subset of NSAIDs (ibuprofen, sulindac
sulfide (SSide), indomethacin, and R-flurbiprofen)
selectively decrease the secretion of A
42 from cultured cells
independently of COX activity and lowers the amount of soluble A
42
in the brains of transgenic mice (21, 22). In cultured cells, the
decrease in A
42 secretion caused by SSide was accompanied by an
increase in A
38 generation, whereas the Notch site-3 cleavage
activity to generate Notch intracellular domain (NICD) was not
significantly affected. In contrast, other NSAIDs including sulindac
sulfone (SSone), which is a metabolite of SSide and inactive for COX,
had no significant effect on
-secretase activity. These data suggest
that some of the NSAIDs may affect the pathogenetic process of AD by
directly inhibiting the
-secretase activity, causing a shift in the
cleavage site. Furthermore, in contrast to the previously developed
-secretase inhibitors, the treatment with NSAIDs may be free from
side effects caused by inhibiting the intramembranous cleavage of other
substrates. However, the molecular mechanism whereby NSAIDs inhibit
A
42 generation, as well as the target protein of NSAIDs to modulate
-secretase activity, still remains unclear. Here we analyzed the
enzymatic property of
-secretase by an in vitro
-secretase assay using recombinant substrates and CHAPSO-solubilized
membrane as an enzyme source, with special reference to the effect of
NSAIDs on intramembranous cleavage of
APP and Notch.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
peptides were purchased from Bachem AG (Bubendorf, Switzerland).
APPNL in pCEP4 (Invitrogen) and Notch
E in
pCS2 were provided by Drs. K. Maruyama (Saitama Medical School) and R. Kopan (Washington University), respectively. A stable Neuro2a (N2a)
cell line doubly expressing
APPNL and Notch
E (N2a
NL/N) was generated as described previously (24). To analyze the effect
of NSAIDs on
-secretase activity, N2a NL/N cells were cultured at
confluency in Dulbecco's modified Eagle's medium containing 10 mM butyric acid to drive protein expression in the presence
of various concentrations of NSAIDs for 48 h. Culture media were
collected and subjected to BAN50/BA27 or BAN50/BC05 ELISAs (25).
Immunoblot analysis using C4 (anti-
APP C terminus, provided from Dr.
Y. Ihara (University of Tokyo)) or anti-c-Myc (Roche Applied Science)
was performed as described previously (8, 9).
APP or 101 amino acids of
mouse Notch 1 fused to FLAG tag at the C terminus and harboring an
additional Met at the N terminus was generated by PCR and subcloned
into pTrcHis2A (Invitrogen) (C100-FmH and N102-FmH, respectively) (26).
A cDNA encoding C100-FmH carrying I716F mutation (11, 27) was
generated by the long PCR protocol using a cDNA encoding C100-FLAG
in pTrcHis2A vector as a template. All constructs were sequenced using
Thermo SequenaseTM (Amersham Biosciences) on an automated
sequencer (Li-Cor). Recombinant proteins were expressed in
Escherichia coli and purified by nickel-chelating affinity
chromatography as described previously (26).
-Secretase Fractions from Cultured
Cells--
All cell lines were maintained as described previously (25)
and grown at confluency. Membrane fractions were prepared as described
previously (9, 28, 29). The membrane pellets were resuspended in HEPES
buffer to yield a protein concentration of 5-10 mg/ml and were stored
at
70 °C. The membranes were solubilized by 1% CHAPSO (Wako,
Osaka, Japan) combined with 1 M NDSB-256 (Calbiochem) for
60 min at 4 °C and centrifuged at 100,000 × g for
60 min. We defined the supernatant (~5 mg of protein/ml) as the
solubilized
-secretase fraction, which was stored at
80 °C
until use. All procedures were performed at 4 °C.
-Secretase Assay--
In vitro
-secretase assay was performed as described previously (26) with
some modifications. Each recombinant substrate at defined
concentrations was incubated together with the solubilized
-secretase fraction (250 µg/ml) in 1×
buffer (HEPES buffer containing 0.25% CHAPSO, 5 mM EDTA, 5 mM
1,10-phenanthroline, 10 mg/ml phosphoramidon, Complete protease
inhibitor mixture (Roche Applied Science)) with or without
-secretase inhibitors (including NSAIDs) at 4 or 37 °C for 3 h. Control reactions were performed in the presence of 1%
Me2SO. The reaction was stopped by boiling the reaction
mixtures for 2 min. The samples were centrifuged, and the supernatants
were analyzed by BAN50/BA27, BAN50/BC05, BNT77/BA27, or BNT77/BC05
ELISAs for de novo generation of A
(25). For the
immunoblot analyses of de novo generated peptides, the total
proteins in the supernatants were precipitated by trichloroacetic acid
and analyzed by immunoblotting with BAN50 (anti-A
), anti-c-Myc (Roche Applied Science), or anti-FLAG M2 (Sigma). Tris/Bicine/urea high
resolution A
immunoblot analysis was performed as described previously (30).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-Secretase Activity in Vitro--
We
first characterized the biochemical and enzymatic properties of the
-secretase activity as detected by our in vitro
-secretase assay. For this purpose, we generated a recombinant
protein substrate C100-FmH, based on the amino acid sequence of the
C-terminal fragment of
APP fused to FLAG-myc-His6 tag
sequences at the C terminus (Fig. 1).
De novo generation of A
peptides from recombinant
C100-FmH incubated with membranes of HeLa cells as an enzyme source
required the presence of 0.25% CHAPSO, whereas Triton X-100 or SDS
abolished the
-secretase activity (data not shown), which was
consistent with the previous observations (12). Incubation of HeLa cell membranes with wild-type (wt) C100-FmH predominantly generated A
40,
in addition to A
42 as a minor species (Fig.
2A). However, the relatively
low levels of de novo generation of A
42 hampered detailed
pharmacological and enzymatic analyses of A
42-generating activity
from wt C100-FmH. Thus, we introduced the I716F mutation into the
recombinant substrate, which had been described to cause a dramatic
increase in A
42 generation in intact cell-based assays (27).
De novo A
42 generation from I716F mutant (mt) C100-FmH was significantly increased, whereas the production of A
40 was almost totally abolished, suggesting that mt C100-FmH served as an
optimal substrate for
42-secretase (Fig. 2A).
These proteolytic activities were recovered from the solubilized
membrane fraction by 1% CHAPSO containing 1 M NDSB-256. We
next examined the effects of the two well characterized
-secretase
inhibitors, L-685,458 and DAPT, on A
generation in our in
vitro assay (12, 23, 31). Both compounds inhibited A
-generating
activities in a similar, concentration-dependent fashion
(Fig. 2B). We then studied the A
-generating activities in
membranes of various cell lines including embryonic fibroblasts derived
from PS1/2 double knockout mice (9, 32). Consistent with the results of
intact cell-based assays, de novo production of A
peptides was almost totally abolished in membrane fractions from PS1/2
double knockout cells, and these activities can be immunoprecipitated
with antibodies against PS1 (Fig. 2C and data not shown).
These data suggest that recombinant wt and mt C100-FmHs were processed
by the endogenous, PS-dependent
40- and
42-secretase activities that enzymatically generate A
40 and -42 polypeptides, respectively. We further analyzed the enzymatic properties of
-secretase activities of intact or
solubilized membrane fractions from HeLa cells in vitro. The
apparent Km value for the processing of either wt or
mt substrate to generate A
40 or A
42, respectively, by
-secretase activities was ~0.5 µM (Fig.
2D). The progress curve was linear during the 6-h reaction time, and the pH dependence of
-secretase activity was broad, ranging from pH 5 to 9 (data not shown). Furthermore, we observed a
-secretase-dependent in vitro generation of
the C-terminally tagged APP intracellular domain fragments (AICD-FmH)
that is produced as the C-terminal counterpart of A
, by incubation
of either wt or mt C100-FmH with solubilized HeLa membranes (Fig.
2E). These data are consistent with the previous reports
(12, 33, 34) on the in vitro
-secretase assays using
different types of recombinant C100 with or without C-terminal
tags.
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Fig. 1.
Schematic representation of recombinant
substrates used in this study. C100-FmH consists of an N-terminal
Met, APP-(597-695) and the consecutive FLAG, c-Myc, and His
tags. Similarly, N102-FmH is composed of Notch-(1699-1799) with an
N-terminal Met and C-terminal FLAG/Myc/His tag. De novo
generated A
peptides and intracellular domains (black box
and white boxes, respectively) were detected by two-site
ELISAs (BAN50/BNT77-BA27/BC05) or using sensitive immunoblotting
methods. The location of I716F mutation in mt C100-FmH is indicated by
star.
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Fig. 2.
Characterization of the in vitro
-secretase activity. A, de novo
generation of A
40 (open columns) or A
42 (filled
columns) peptides from recombinant substrates in vitro
using HeLa cell membranes as enzyme source and quantitated by ELISAs.
Names of recombinant proteins used as substrates are indicated
below the columns. B, inhibitory potencies of
solubilized
-secretase activity by DAPT (left panel) and
L-685,458 (right panel). De novo generated levels
of A
40 from wt substrate (open circles) or A
42 from mt
substrate (filled triangles) were evaluated by ELISAs. The
data are indicated as % of those observed in the absence of given
inhibitors. C, generation of A
40 from wt (open
columns) or A
42 from mt (filled columns) substrates
by membrane fractions from various cell lines. CHO, Chinese
hamster ovary; MDCK, Madin-Darby canine kidney.
D, Michaelis-Menten plot for the generation of A
40 from
wt substrate (open circles) or A
42 from mt substrate
(filled triangles). E, AICD-FmH generation from
recombinant substrates in vitro. Total proteins in the
samples incubated overnight at the indicated temperature (4 or
37 °C) in the presence or absence of DAPT were precipitated by
trichloroacetic acid and analyzed by immunoblotting. Recombinant
C100-FmH and the cleavage product thereof (AICD-FmH) are indicated by
the arrowhead and arrow, respectively.
F, generation of NICD-FmH from recombinant substrates
in vitro as in E. Recombinant N102-FmH and
cleavage products (NICD-FmH) are indicated by the arrowhead
and arrow, respectively.
-secretase substrates generates the
intracellular domain fragments that are liberated from the membrane and
mediate the signal pathways from plasma membrane to nucleus (2).
Notably, a proteolytic generation of Notch intracellular domain (NICD)
by PS-dependent
-secretase is the most well known
example, and potential side effects caused by the blockade of Notch
pathway by
-secretase inhibitors are emerging problems (35, 36). To
analyze the
-secretase activity to generate NICD in
vitro, we generated a recombinant N102-FmH substrate composed of a
101-residue fragment of murine Notch1 beginning close to the S2
cleavage site and containing transmembrane domain fused to a
FLAG-myc-His C-terminal tag (Fig. 1). After incubation of N102-FmH with
the membrane fraction, we observed the appearance of an NICD-like
polypeptide migrating slightly faster than N102-FmH, which was
diminished by treatment with DAPT in a dose-dependent manner (Fig. 2F and data not shown). Thus, the recombinant
N102-FmH polypeptide is also cleaved by
-secretase to generate
C-terminally tagged NICD (NICD-FmH) in vitro, which was
consistent with the recent report by Wolfe and colleagues (37, 38).
-Secretase Activity in
Vitro--
It has been reported recently (21, 22) that a subset of
NSAIDs lower A
42 without affecting Notch processing in cultured cells. To gain more insights into the effect of NSAIDs on APP and Notch
processing, we have chosen three NSAIDs, i.e. sulindac sulfide (SSide), sulindac sulfone (SSone), and naproxen, to treat N2a
NL/N cells stably coexpressing
APPNL and Notch
E (24). We confirmed a specific decrease in A
42 secretion by treatment with
10-30 µM SSide, whereas the secretion of A
40 as well
as Notch processing was not affected (Fig.
3). Treatment with 100 µM
of SSide caused cell death presumably by inducing apoptosis, resulting
in marked decrease in A
generation as well as in total protein
expression (17). The IC50 value for A
42 secretion of SSide was 30.6 ± 2.8 µM. SSone and naproxen had no
effect either on A
40 or A
42 secretion as well as on Notch
cleavage up to 100 µM.
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Fig. 3.
Inhibitory effects of NSAIDs on
A secretion and Notch processing in N2a NL/N
cells. A, ELISA quantitation of A
40 (open
circles) and A
42 (filled triangles) secretion from
N2a NL/N cells in the presence of NSAIDs at the indicated
concentrations. The data are indicated as % of those observed in the
absence of inhibitors. B, immunoblot analysis of the
expression and processing of
APP and Notch in N2a cell lysates
treated by NSAIDs at the indicated concentrations. Cell lysates were
separated by SDS-PAGE and analyzed by immunoblotting with anti-
APP
antibody C4 (upper and middle
panel) or anti-c-Myc (lower panel),
respectively. Protein bands corresponding to
APPNL or
Notch
E and their cleavage products (C83 or NICD, respectively) are
indicated by arrows and arrowheads,
respectively.
-secretase activity by direct or
indirect mechanisms (e.g. altering the trafficking of substrates or enzymes, affecting the secretion or degradation of A
42
peptides, or modifying the transcription of
-secretase-related genes), we analyzed the in vitro
-secretase activity in
solubilized membrane fraction in the presence of NSAIDs. We observed an
inhibition of
42-secretase activity by SSide in a
dose-dependent manner. The IC50 value of SSide
for inhibiting
42-secretase activity in vitro
was 20.2 ± 2.6 µM (Fig.
4A). We found a decrease in
slope by the increase of the concentration of SSide in the plot of rate against the enzyme concentration, suggesting that SSide is not an
irreversible or pseudo-irreversible inhibitor (Fig. 4B).
Moreover, when we dialyzed the solubilized
-secretase fraction
pretreated with SSide against CHAPSO buffer without SSide,
-secretase activity was almost totally recovered (Fig.
4C). From these data, it was strongly suggested that the
genuine molecular target of SSide is the
-secretase complex and that
SSide works as a reversible
-secretase inhibitor.
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Fig. 4.
Inhibitory effects of NSAIDs on in
vitro -secretase activity. A, ELISA quantitation of
de novo generated A
40 (open circles) and
A
42 (filled triangles) from recombinant substrates in the
presence of NSAIDs at the indicated concentrations. The data are
indicated as % of those generated in the absence of inhibitors. Note
that SSide, but not SSone or naproxen, inhibits both
40-
and
42-secretase activity at high concentrations.
B, plot of activity rate against the enzyme concentration
under coincubation with 0 (open circles), 25 (filled
circles), 50 (open squares), and 75 µM (filled squares) SSide. C,
de novo A
42 generation by dialyzed membrane fraction
preincubated with 100 µM SSide. Note that the dialysis of
SSide-preincubated
42-secretase resulted in the almost
total recovery of the enzymatic activity.
40 generation in vitro (Fig.
4A). Moreover, SSide diminished the de novo
generation of A
40, in addition to that of A
42, at high
concentrations (50-100 µM). Thus, SSide has an
inhibition potency against
40-secretase activity at high
concentrations, whereas it elevates the
40-secretase activity at sub-inhibitory doses. It was reported that the decrease in
A
42 secretion by SSide was accompanied by a
dose-dependent increase in A
38 secretion (21). To
determine whether the decrease in A
42 (plus A
40) production
caused by high concentrations of SSide affects that of A
38 in
vitro, we analyzed the de novo generated A
species
by high resolution immunoblotting (30). We observed a
dose-dependent increase in A
38 generation in the low
concentration ranges (1-25 µM) accompanied by a decrease
in A
42 production, although the in vitro generation of
A
peptides including A
38 was entirely inhibited by high
concentrations of SSide (data not shown). These results suggest that
SSide is a bona fide
-secretase inhibitor directly
affecting the membrane-embedded protease complex, exhibiting distinct
inhibitory potencies against A
38-, A
40-, and A
42-generating
activities of
-secretase.
42-secretase activity by SSide, we performed the
double-reciprocal plot analysis (Fig.
5A). We found that the
Km value remained at a constant level, but the
Vmax value was decreased under increasing concentrations of SSide, suggesting that SSide displayed a
noncompetitive inhibition for
42-secretase activity.
Because transition state analogue
-secretase inhibitors
(i.e. pepstatin or L-685,458) displayed linear
noncompetitive inhibition profiles, a two-binding site model for
intramembranous cleavage by
-secretase has been proposed (34). In
this model,
-secretase complex is predicted to harbor a
docking/anchoring site of substrates as well as a separate catalytic
site. To determine whether SSide affects
42-secretase activity by interacting with the catalytic site of
42-secretase, we analyzed the inhibition profile of
SSide by coincubation with L-685,458, a transition state analogue
inhibitor that is expected to occupy the active site of
42-secretase. If SSide binds to the docking/anchoring
site, coincubation with SSide and L-685,458 would result in a
synergistic inhibition of
42-secretase and cause an
increase in the inhibition slope. Unexpectedly, however, an addition of
L-685,458 had no effect on the slope, raising the possibility that
SSide may inhibit the
42-secretase activity in a similar
mechanism to that of a transition state analogue inhibitor, L-685,458
(Fig. 5B). Thus, SSide would compete for a similar binding
site (i.e. catalytic site) with L-685,458 in the
42-secretase complex or, alternatively, the binding of
either of the inhibitors allosterically affects the interaction of the other.
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Fig. 5.
Characterization of the inhibitory effect of
SSide on 42-secretase. A, double-reciprocal
plots for inhibition of
42-secretase by 0 (open
circles), 25 (filled circles), 50 (open
squares), 75 (filled squares), and 100 µM
(open triangles) SSide. The data are indicated as % of
those observed in the absence of SSide. B, intercept
replot of inhibition of
42-secretase by SSide in the
presence of 0 (open circles), 0.5 (filled
circles), 1 (open squares), and 2 nM
(filled squares) L-685,458. The data are indicated as % of
those generated in the absence of inhibitors.
APP and Notch in vitro (Fig. 6). We observed that the inhibition
kinetics of AICD-FmH generation from C100-FmH by SSide was
approximately similar to that of the A
generation; the proteolytic
activity to release AICD-FmH from wt C100-FmH was increased by
treatment with 10-25 µM SSide, whereas it was completely
inhibited at 100 µM. In contrast, AICD-FmH production
from mt C100-FmH was inhibited by SSide in a dose-dependent
fashion at 10-100 µM. We then analyzed the effect of
SSide on NICD-FmH generation from recombinant substrate in vitro. The endoproteolytic cleavage of N102-FmH was inhibited by
high concentrations of SSide (250-500 µM), whereas it
was not affected by SSide at concentrations up to 100 µM,
which was consistent with the results obtained in cultured cells (Fig.
3B). These results demonstrate that SSide can inhibit the
-secretase activity for Notch as well as for
APP, although its
inhibition potency for Notch is much weaker than that for
APP,
especially for
42-cleavage.
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Fig. 6.
Characterization of the inhibitory effect of
SSide on intracellular domain generation of
APP and Notch in vitro.
A-C, immunoblot analysis of de novo generation
of AICD-FmH (arrow in A or B) or
NICD-FmH (arrow in C) in the presence of SSide or
SSone. The names of substrates used in the experiments are indicated
below the lanes. D, densitometric analysis of the
levels of AICD-FmH from wt substrate (open circles),
AICD-FmH from mt substrate (filled triangles), and NICD-FmH
(shaded squares). The averages of intensities of each band
in two independent experiments are shown.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
42, although the molecular mechanism whereby NSAIDs
affect the
-secretase activity remained unclear (21, 22). In this
study, we established an in vitro
-secretase assay using
recombinant wild type as well as mutant C100 as substrates and analyzed
the effect of NSAIDs. We found that SSide, but not its metabolite SSone
nor naproxen, directly inhibits the
-secretase activity derived from
membrane fractions of HeLa cells in a dose-dependent manner. Moreover, we showed that SSide is a bona fide
-secretase inhibitor that has the highest inhibition potency against
A
42-cleaving activity compared with those for A
38, A
40, or
Notch, with noncompetitive inhibition kinetics.
B kinase
activity, activate PPAR
,
inactivate PPAR
, inhibit Ras signaling, and reduce the proliferation
and induce apoptosis of cancer cells (17). It has been shown that the
A
42-lowering effect of NSAIDs is independent of COX-inhibiting
activity in cultured cells (21). We confirmed the A
42-specific
inhibition of A
secretion in culture cells by SSide, although the
analysis of the inhibition profile at high concentrations was difficult
because of the cell toxicity. To examine whether SSide directly
inhibits
-secretase, we employed an in vitro assay system
using solubilized membranes as enzyme sources and recombinant C100
polypeptides as substrates, and we demonstrated that SSide has the
capacity to inhibit the total
-secretase activity, although cleavage
at A
42 site was most effectively inhibited. In addition, in
vitro A
generation took place in the absence of any NTPs,
suggesting that kinase activities (e.g. I
B kinase
)
are not involved in the regulation of
-secretase by SSide; we also
observed that addition of ATP does not change the level of de
novo A
generation, suggesting that
-secretase activity does
not require energy.2 Finally,
we showed that SSide displayed noncompetitive inhibition kinetics
in vitro, which is a common characteristic of a number of
-secretase inhibitors (34). From these data, we postulate that SSide
works as a
-secretase inhibitor that directly affects its activity
by binding to the membrane-embedded protease complex.
-secretase inhibitors have been documented to
inhibit secretion of A
40 and A
42 in two different patterns. The
peptide aldehydes and peptidomimetic inhibitors containing a
difluoroketone or alcohol group increase A
42 secretion at
sub-inhibitory doses and diminish it at a high concentration, whereas
they inhibit A
40 generation in an absolutely
dose-dependent manner (39-41). However, the rank order of
inhibitory potencies of several peptide aldehydes against A
40 and
A
42 are at similar levels, suggesting that a single
-secretase
complex would generate A
40 and A
42 (40). In addition, the
transition state analogue inhibitors of aspartyl proteases containing a
hydroxyethylene isostere also inhibited the secretion of A
40 and
A
42 to similar extents (12, 41). The compounds display similar
inhibition kinetics between a cell-free system (i.e.
incubation of the membrane fraction that harbors both enzyme and
substrate) and an in vitro assay using recombinant
substrates, suggesting that these compounds act directly on
-secretase and that the differences in the inhibition kinetics might
depend on their binding sites or target molecule(s) within the
-secretase complex.
40 and A
42 generation at low concentrations of peptide aldehyde
inhibitors and SSide still remains unknown. One possible explanation is
that a partial loss of
40-secretase function by the low
concentrations of peptide aldehydes or difluoroketone peptide mimetics
would shift the substrate supply to
42-secretase that is
still active, thereby leading to overproduction of A
42. In sharp
contrast, SSide exhibited entirely novel inhibition profiles to
preferentially inhibit A
42 generation, which was accompanied by an
increase in the production of A
38 as well as A
40 at
sub-inhibitory concentration ranges in vitro. We speculate
that SSide may act on a component that is distinct from those affected
by peptide aldehydes or difluoroketone protease inhibitors.
Alternatively, SSide and other inhibitors may exert opposite effects on
a component that is involved in the determination of the position of a
scissile bond to be cleaved in
-secretase complex. Unexpectedly, a
coincubation study with the transition state analogue inhibitor,
L-685,458, showed a direct competition with SSide, raising the
possibility that SSide might directly act on the catalytic site,
although the structure of SSide is not similar to any known substrates. An alternative possibility would be that SSide binds to the
noncatalytic site of
-secretase complex and allosterically regulates
the catalytic site in a way to dissociate substrates and active
site-specific inhibitors, showing an apparent direct competition. Such
reciprocal regulation of different proteolytic activities by protease
inhibitors or substrates has been observed in proteasome that harbors
three distinct proteolytic activities (i.e.
chymotrypsin-like, trypsin-like, and peptidylglutamyl
peptide-hydrolyzing activities) (42-44). Ritonavir, an inhibitor of
human immunodeficiency virus-1 protease, competitively inhibits the
chymotrypsin-like activity, whereas trypsin-like activity is enhanced
(43). Extensive studies using active site-specific inhibitors suggested
that proteasome effectors/substrates (e.g. ritonavir) that
cause reciprocal regulation might act on noncatalytic sites, rather
than through binding to an active site. Further studies using
derivatives of SSide that contain affinity moiety (e.g.
photoreactive groups) are needed to obtain definitive proof that SSide
acts directly on
-secretase.
-secretase inhibitors
abolish the site-3 cleavage of Notch in cultured cells, with the
exception of SSide and a nonpeptidic isocoumarin derivative, JLK6 (21,
45). However, it has been shown that JLK6 fails to inhibit
-secretase activity in vitro, suggesting that JLK6 is not
a direct inhibitor of
-secretase (46). We found that SSide has the
capacity to inhibit the endoproteolysis of Notch at the S3 site at much
higher concentrations compared with those for A
inhibition in
vitro, whereas other peptidic inhibitors (e.g. DAPT)
abolished Notch cleavage with similar potencies to those for A
generation. These results suggest that
- and Notch secretases are
pharmacologically distinct but related. Thus, it may be possible to
avoid the envisaged side effects of
-secretase inhibitors caused by
inhibition of Notch signaling by developing derivatives of SSide.
Further attempts to define the molecular mechanisms of inhibition on
-secretase activity by SSide and to screen its derivatives
specifically relevant to
APP cleavage will facilitate not only the
development of a novel therapeutic drug for AD but also our
understanding of the unusual intramembranous proteolytic activity of
-secretase that cleaves membrane-spanning proteins at multiple positions.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Drs. Y. Ihara, B. De Strooper, and
R. Kopan for providing antibody, mouse embryonic fibroblasts
lacking PS1/2 and NE cDNA, respectively, and Takeda
Chemical Industries for continuous support for our studies.
![]() |
FOOTNOTES |
---|
* This work was supported by grants-in-aid from the Ministry of Health and Welfare, and the Ministry of Education, Science, Culture and Sports for the 21st Century Center of Excellence Program, Japan.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.
To whom correspondence may be addressed: Dept. of
Neuropathology and Neuroscience, Graduate School of Pharmaceutical
Sciences, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. Tel.: 81-3-5841-4868; Fax: 81-3-5841-4708; E-mail:
taisuke@ mol.f.u-tokyo.ac.jp.
** To whom correspondence may be addressed: Dept. of Neuropathology and Neuroscience, Graduate School of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. Tel.: 81-3-5841-4877; Fax: 81-3-5841-4708; E-mail: iwatsubo@mol. f.u-tokyo.ac.jp.
Published, JBC Papers in Press, March 10, 2003, DOI 10.1074/jbc.M301619200
2 T. Tomita, Y. Takahashi, and T. Iwatsubo, unpublished observations.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
AD, Alzheimer's
disease;
A, amyloid
peptide;
APP, amyloid-
precursor
protein;
CHAPSO, 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate;
COX, cyclooxygenase;
DAPT, N-[N-(3,5-difluorophenacetyl)-L-alanyl]-(S)-phenylglycine
t-butyl ester;
ELISA, enzyme-linked immunosorbent assay;
NICD, Notch intracellular domain;
NSAIDs, nonsteroidal
anti-inflammatory drugs;
mt, mutant;
PS, presenilin;
SSide, sulindac
sulfide;
SSone, sulindac sulfone;
wt, wild-type;
Bicine, N,N-bis(2-hydroxyethyl)glycine;
HMW, high
molecular weight;
AICD, APP intracellular domain;
PPAR, peroxisome
proliferator-activated receptors.
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