From the Institute of Biochemistry, Swiss Federal Institute of Technology (ETH), ETH-Hoenggerberg, 8093 Zurich, Switzerland and § Nervous System Research, Novartis Pharma AG, 4002 Basel, Switzerland
Received for publication, February 7, 2003
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ABSTRACT |
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Presenilin is implicated in the
pathogenesis of Alzheimer's disease. It is thought to constitute the
catalytic subunit of the Alzheimer's disease
(AD)1 is characterized by the
formation of senile plaques in the brain. Major constituents of
these plaques are the amyloidogenic 40- and 42-residue-long A To date, the majority of characterized familial AD mutations are
clustered along the presenilin-1 (PS1) gene (10, 11). They are thought
to accelerate disease onset by increasing the A Recently, we identified the intramembrane-cleaving protease SPP (for
signal peptide peptidase) that contains motifs YD and LGLGD
characteristic for GXGD aspartic proteases (29).
These identical motifs are present in the predicted transmembrane
regions of PS1, supporting its function as an intramembrane-cleaving
aspartic protease and hence a catalytic subunit of the
The common features of SPP and PS1 raise the question of whether
Synthesis of Inhibitors--
L-658,485 (34), L-852,646 (19),
DAPT (WO 9822494), LY411575 (WO 9828268), (Z-LL)2-ketone
(35), and TBL4K (29) were synthesized as described
previously. NVP-AHW700-NX was synthesized according to methods reported
for a related compound (36). The purity of each compound was checked by
1H nuclear magnetic resonance (NMR) spectroscopy, mass
spectroscopy, high-pressure liquid chromatography, and thin-layer
chromatography, and the results were consistent with the expected
structures. JLK2 (37) was kindly provided by F. Checler and
pepstatin A was purchased from Sigma.
For testing SPP Assay and Affinity Labeling--
Inhibition of SPP in Tissue Culture Cells and Indirect
Immunofluorescence--
Hepatitis C virus structural proteins C, E1,
and E2 were transiently expressed in baby hamster kidney C13 cells as
described previously (33). Following electroporation with in
vitro transcribed mRNA encoding the CE1E2 polyprotein, cells
were diluted in growth medium at a concentration of ~106
cells/ml. An 0.25-ml cell suspension was diluted with 0.25 ml of growth
medium containing either 2% Me2SO, or 2% 100×
concentrated inhibitor dissolved in Me2SO and seeded in
24-well tissue culture plates. After incubation at 37 °C for 10 h, cells were either solubilized in SDS-PAGE sample buffer or fixed for
indirect immunofluorescence analysis with monoclonal core-specific
antibody JM122 (43) (gift from J. McLauchlan) and staining of lipid
droplets (43). For Western blot analysis, proteins were first separated
by SDS-PAGE using 13% polyacrylamide Tris-glycine gels, transferred to
polyvinylidene difluoride membranes, and probed with polyclonal
core-specific antibody R308 (43) (gift from J. McLauchlan). Bound
antibody was detected by enhanced chemiluminescence.
Introducing
The latter finding was confirmed in a cell-free in vitro
assay using detergent-solubilized HEK cell membranes containing
Inhibition of Detergent-solubilized SPP--
We next investigated
the effect of Active Site Labeling of SPP and Competition with
To further demonstrate that some of the Potency of
As depicted in Fig. 4A, (Z-LL)2-ketone,
L-685,458, NVP-AHW700-NX, and LY411575 inhibited the processing of HCV
core protein. Apparent IC50 values varied from ~10
nM (for LY411575) to ~5 µM (for L-685,458).
The IC50 values observed with the less membrane-permeable compounds, (Z-LL)2-ketone and L-685,458, were much higher
than in the in vitro assays. These compounds most likely
penetrate the plasma membrane to a lower extent compared with the less
peptidic and therefore more permeable compounds LY411575 and
NVP-AHW700-NX, which showed comparable IC50 values in both
assays. DAPT and pepstatin A did not inhibit the processing of HCV core
protein and hence did not affect SPP, as already observed in the
cell-free in vitro assay. Also JKL2 did not affect the
processing of HCV core protein at concentrations up to ~10
µM, at which level the compound started to become
cytotoxic (not shown).
The consequences of SPP inhibition on the processing of HCV core
protein were next visualized by indirect immunofluorescence. When
processed and released from the ER membrane, core protein was found
associated at the surface of lipid droplets in the cytosol and appeared
in characteristic ring-like structures (Fig. 4B). When
expressed in the presence of (Z-LL)2-ketone, L-685,458,
NVP-AHW700-NX, and LY411575, all of which inhibit SPP, HCV core protein
did not localize to lipid droplets and appeared in a reticular staining pattern, indicating retention in the ER membrane. DAPT and pepstatin A,
which do not affect the processing of HCV core protein, had also no
effect on its intracellular distribution. Taken together, (Z-LL)2-ketone and the In the present study we have demonstrated that aspartic protease
inhibitors directed against The nature of the catalytic site of the Additional indirect evidence that PS1 is a protease was provided by the
present study reporting on overlapping inhibitor activities. Compounds,
including transition state analogues, were found to efficiently inhibit
both Although three compounds, pepstatin A, DAPT, and
(Z-LL)2-ketone, could discriminate between
SPP and -secretase complex that catalyzes
intramembrane cleavage of
-amyloid precursor protein, the last step
in the generation of amyloidogenic A
peptides. The latter are major
constituents of amyloid plaques in the brain of Alzheimer's disease
patients. Inhibitors of
-secretase are considered potential
therapeutics for the treatment of this disease because they prevent
production of A
peptides. Recently, we discovered a family of
presenilin-type aspartic proteases. The founding member, signal peptide
peptidase, catalyzes intramembrane cleavage of distinct signal peptides
in the endoplasmic reticulum membrane of animals. In humans, the protease plays a crucial role in the immune system. Moreover, it is
exploited by the hepatitis C virus for the processing of the structural
components of the virion and hence is an attractive target for
anti-infective intervention. Signal peptide peptidase and presenilin
share identical active site motifs and both catalyze intramembrane
proteolysis. These common features let us speculate that
-secretase
inhibitors directed against presenilin may also inhibit signal peptide
peptidase. Here we demonstrate that some of the most potent known
-secretase inhibitors efficiently inhibit signal peptide peptidase.
However, we found compounds that showed higher specificity for one or
the other protease. Our findings highlight the possibility of
developing selective inhibitors aimed at reducing A
generation
without affecting other intramembrane-cleaving aspartic proteases.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
peptides A
40 and A
42, respectively (1). The amyloid cascade
hypothesis casually links the generation of amyloid plaques with the
neuropathological changes accompanying the symptoms typical of
this disease (2). A
peptides are generated from the type I
transmembrane protein
-APP (
-amyloid precursor protein) by
sequential proteolysis (3). The protein is first cleaved in the
exoplasmic domain by the
-site APP-cleaving enzyme (BACE) to release
the ectodomain (4, 5). The residual membrane-anchored stub of 99 residues (C99) is subsequently cleaved in the center of the
transmembrane region by
-secretase (6). The resulting cleavage
products, an A
peptide and the amyloid intracellular domain (AICD),
are liberated from the lipid bilayer toward the exoplasm and cytosol, respectively (7-9).
42/A
40 ratio (12).
It is not well understood how these mutations, which are essentially
scattered along the entire PS1 gene, can lead to a specific increase in
the production of the 42-residue-long peptide that corresponds to the
most amyloidogenic form of A
(13). It has been shown that PS1 plays
a key role in transport and maturation of
-APP (14). It is also an
essential component of the
-secretase complex (6), and several lines
of evidences suggest that PS1 may constitute the catalytic subunit of
this multi-subunit protease (15). For example, several aspartic
protease transition state analogues have been found to inhibit
-secretase activity and target PS1 (16-20), and conservative
mutations of putative active site aspartates in PS1 result in the loss
of
-secretase activity (21, 22). Thus, in recent years, the
development of small molecular weight compounds aimed at reducing
-secretase/PS1 activity as a possible therapeutic strategy for AD
has attracted major attention. Several potent inhibitors that affect
-secretase/PS1 in cellular assays have been reported, and at least
one compound has been shown to reduce plaque load in a transgenic
animal model for AD-type amyloidosis (23). The major concern related to
this approach is that
-secretase/PS1 not only catalyzes the
processing of C99, but it is also required for the processing of other
transmembrane proteins such as CD44 (24), the tyrosine kinase receptor
Erb4 (25, 26), and the Notch receptor family (27, 28).
-secretase/PS1 complex (6, 30). SPP promotes intramembrane
proteolysis of distinct signal peptides after they have been cleaved
off from newly synthesized secretory or membrane proteins in the
endoplasmic reticulum (ER) membrane of higher eukaryotes (29, 31). In humans, SPP is essential for the generation of signal sequence-derived human lymphocyte antigen (HLA)-E epitopes and thus plays a crucial role
in our immune system (32). Furthermore, SPP promotes cleavage at an
internal signal sequence in the hepatitis C virus (HCV) polyprotein and
is essential for proper maturation of the viral core protein (33).
Inhibitors of SPP may thus be considered as potential therapeutics for
the treatment of HCV infection.
-secretase/PS1 inhibitors directed against the putative active site
of PS1, for example aspartic protease transition state analogues, are
also acting against SPP and hence affect intramembrane-cleavage of
signal peptides. In the present study, we investigated the effects of
representative, potent
-secretase/PS1 inhibitors on SPP activity. We
first tested the compounds for their potency in blocking A
generation in intact cells as well as inhibiting solubilized
-secretase activity in a cell-free in vitro assay. In the
same type of assays, we then investigated the effect of these compounds
on SPP activity and assessed their propensity to compete with active
site labeling.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Secretase Assays--
Inhibition of
-secretase activity
in live cells was assayed by quantifying the generation of secreted
A
. In brief, human embryonic kidney cells (HEK)-293 cells stably
transfected with
-APP carrying the Swedish mutation (38,
39) were plated in microtiter plates. After 1 day, the inhibitors were
added in fresh medium, and the cells were incubated for another 24 h. 10 µl of conditioned medium were removed for determination of A
levels by sandwich enzyme-linked immunosorbent assay using the
A
40-specific monoclonal antibody 25H10 raised against the free
C-terminal peptide, MVGGVV, of A
40. The monoclonal
1
antibody (39) was biotinylated and used as a detection antibody with
alkaline phosphatase coupled to streptavidin. For chemiluminescence,
substrate CSPD (disodium 3-(c-methoxyspiro{1,2-dioxetane-3,2'-(5'-chloro)tricyclo[3.3.1.13,7]decan}-4-yl)phenyl phosphate) and the enhancer EmeraldII (Tropix) were applied.
Standard curves with synthetic A
40 peptide (Bachem) were run in parallel.
-secretase in vitro, detergent-solubilized
-secretase activity was prepared from HEK-293 cells (40) and
incubated with substrate Met-C99, which was synthesized by in
vitro translation (see below), and either Me2SO
(2%) or inhibitor at the indicated concentration. After incubation,
samples were subjected to immunoprecipitation with antibody 25H10 and
analyzed by SDS-PAGE and phosphorimaging using 15% polyacrylamide
Tris-Bicine-urea acrylamide gels (41) and a STORM PhosphorImager
(Amersham Biosciences). Reference peptide Met-A
40 was synthesized by
in vitro translation.
-Secretase inhibitors
were tested on SPP in a previously established in vitro
assay (35). In brief, 2 µl of cell-free translation mixture
containing [35S]methionine-labeled peptide
p-PrlPP29/30 (31) were diluted with 35 µl of
SPP buffer (25 mM HEPES-KOH, pH 7.6, 100 mM
KOAc, 2 mM Mg(OAc)2, 1 mM
dithiothreitol) and supplemented with 1 µl of 100× concentrated
inhibitor in Me2SO. Reactions were initiated by the
addition of 2 µl of CHAPS-solubilized ER membrane proteins, and
samples were incubated for 1 h at 30 °C. Samples were analyzed
next by SDS-PAGE and phosphorimaging using 15% polyacrylamide Tris-Bicine-urea acrylamide gels (41) and a STORM PhosphorImager (Amersham Biosciences). Quantification was performed with IQMac version
1.2 software (Amersham Biosciences). For affinity labeling, CHAPS-solubilized ER membrane proteins were incubated in SPP buffer in
the presence of 50 nM TBL4K or 25 nM L-852,646 and the indicated concentrations of competitor
(29). Samples were incubated at 30 °C for 30 min and subsequently
irradiated with UV light (30 s for TBL4K, 5 min for
L-852,646; 350-watt high pressure mercury lamp, 10-cm distance
to lamp) (29). Samples were analyzed by SDS-PAGE on 12% polyacrylamide
Tris-glycine gels (42), and biotinylated proteins were visualized by
enhanced chemiluminescence (Amersham Biosciences) after Western
blotting with a polyclonal anti-biotin antibody (Bethyl) (29).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Secretase/PS1 Inhibitors and Potency of
SPP Inhibitor against
-Secretase/PS1--
The potency of
-secretase/PS1 inhibitors L-685,458 (18), L-852,646 (19), and DAPT
(WO 9822494), second generation compounds LY411575 (WO 9828268), a more
potent analogue of DAPT, and a novel compound NVP-AHW700-NX, a
derivative of L-685,458, as well as the SPP inhibitors
(Z-LL)2-ketone (35) and TBL4K (29) were investigated in this study (Fig.
1A). In a first series of
experiments, we tested whether NVP-AHW700-NX and
(Z-LL)2-ketone function as
-secretase/PS1 inhibitors and
affect generation of A
peptides and compared the potency of the two
compounds with known
-secretase/PS1 inhibitors DAPT, L-685,458, and
LY411575 (Fig. 1B). Stably transfected HEK-293 cells
expressing
-APP were treated with various concentrations of
inhibitor. Following incubation for 24 h, medium was removed and
analyzed for A
peptides in a sandwich enzyme-linked immunosorbent assay. Compounds LY411575 and NVP-AHW700-NX efficiently inhibited A
generation with IC50 values of 0.4 nM and 0.62 µM, respectively, as well as the previously described
inhibitors L-685,458 (0.46 µM) and DAPT (0.17 µM). In contrast, (Z-LL)2-ketone did not
inhibit the generation of soluble A
40 up to a concentration of 100 µM.
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Fig. 1.
Inhibitory potency of
-secretase/PS1 inhibitors and
(Z-LL)2-ketone. A, chemical
structure of inhibitors and photoaffinity labels used in this study.
B, inhibition of
-secretase/PS1 in live cells. Amyloid
precursor protein carrying the Swedish mutation was
expressed in HEK cells in the presence of indicated inhibitors.
IC50 values were determined by measuring the levels of
A
40 secreted into the medium. C, effect of
(Z-LL)2-ketone on detergent-solubilized
-secretase/PS1
activity. Radiolabeled
-secretase/PS1 substrate Met-C99 was added to
CHAPSO-solubilized HEK cell membranes and incubated in the
presence of 5 µM DAPT or 100 µM
(Z-LL)2-ketone. Samples were immunoprecipitated with
A
40-specific antibody. ref, reference peptide
Met-A
40.
-secretase/PS1 activity (Fig. 1C). As a substrate, we
used Met-C99, which was synthesized by cell-free in vitro
translation. This peptide corresponded to the natural substrate of
-secretase/PS1, C99, with an additional N-terminal methionine
required to initiate peptide synthesis. After incubation, samples were
subjected to immunoprecipitation with an A
40-specific antiserum and
analyzed by SDS-PAGE and phosphorimaging. As expected, the
-secretase/PS1 inhibitor DAPT (5 µM) blocked the
generation of A
40. The SPP inhibitor (Z-LL)2-ketone, in
contrast, did not affect production of A
40 up to a concentration of
100 µM.
-secretase/PS1 inhibitors on SPP activity, first in a
previously described cell-free in vitro assay (35). A
radiolabeled SPP substrate, peptide p-PrlPP29/30
(31), was prepared by cell-free in vitro translation in wheat germ extract and incubated with detergent-solubilized ER membrane
proteins containing SPP. Cleavage of the 30-residue-long p-PrlPP29/30 by SPP resulted in the generation
of an ~20-residue-long product that was readily detected and
quantified by SDS-PAGE and phosphorimaging (Fig.
2). The addition of the SPP inhibitors
(Z-LL)2-ketone and TBL4K and the
-secretase/PS1 inhibitors L-685,458, L-852,646, LY411575, and
NVP-AHW700-NX efficiently inhibited cleavage of p-PrlPP29 with apparent IC50 values
ranging from 8 to ~100 nM. Interestingly, the
-secretase/PS1 inhibitor DAPT, which is a less potent derivative of
LY411575, had no effect on SPP activity at concentrations up to 100 µM (Fig. 2). Also, pepstatin A and JKL2, both of which were reported to inhibit
-secretase/PS1 activity (37, 40), did not
affect SPP at concentrations up to 100 µM.
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Fig. 2.
Inhibition of detergent-solubilized SPP
with -secretase/PS1 inhibitors.
Radiolabeled SPP substrate p-PrlPP29/30
(SP/30) was incubated with detergent-solubilized SPP
activity in the presence of inhibitor at the indicated concentrations.
Samples were analyzed by SDS-PAGE and phosphorimaging (example is shown
for L-685,458). For quantification of signal peptide processing, the
amount of cleavage product (SP/20) obtained in the presence
of inhibitor is expressed as percent of that obtained without
inhibitor. L-685,458, filled squares; L-852,646,
filled circles; DAPT, open triangles; LY411575,
filled triangles; NVP-AHW700-NX, filled diamonds;
(Z-LL)2-ketone, asterisks; TBL4K,
crosses; pepstatin A, open diamonds; JKL2,
open circles.
-Secretase/PS1 Inhibitors--
To test whether the
effective
-secretase/PS1 inhibitors affect SPP by binding to the
active site of SPP, we labeled the protease with the previously
described photoaffinity label, TBL4K (29) in the presence
of increasing amounts of inhibitors (Fig.
3A). The central ketone moiety
of TBL4K, a derivative of (Z-LL)2-ketone, is
thought to be converted in situ to a transition state
mimicking gem-diol upon binding to the SPP active site. As expected,
increasing concentrations of the transition state analogues L-685,458
and NVP-AHW700-NX progressively displaced TBL4K from SPP
(Fig. 3A). Likewise, the most potent
-secretase/PS1
inhibitor tested, LY411575, reduced labeling of SPP in a
dose-dependent manner. In agreement with what we observed
in the cell-free in vitro SPP assay, DAPT (Fig.
3A), pepstatin A, and JKL2 (not shown) did not influence the
labeling of SPP.
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Fig. 3.
Photoaffinity labeling of SPP and competition
with -secretase/PS1 inhibitors.
A, labeling with TBL4K and competition. SPP was
labeled with TBL4K in the presence of
-secretase/PS1
inhibitors L-685,458, NVP-AHW700-NX, LY411575, and DAPT at the
indicated concentrations. B, labeling with L-852,646 and
competition with the SPP inhibitor (Z-LL)2-ketone. No
labeling of SPP was observed in the controls without activation of the
reagent (no UV) and in the absence of label (no
Label). The high molecular mass band observed in all lanes
corresponds to a biotinylated protein present in the solubilized
material.
-secretase/PS1 inhibitors
target SPP, we made use of the photoreactive compound L-852,646, a
derivative of L-685,458, that was applied previously to label PS1 in
detergent-solubilized HeLa total cell membranes (19). When incubated
with detergent-solubilized ER membrane proteins and activated with UV
light, L-852,646 selectively labeled an ~40-kDa protein such as
TBL4K (Fig. 3B). The addition of increasing amounts of the SPP inhibitor (Z-LL)2-ketone progressively
reduced labeling. Consistently, compounds that inhibited SPP in the
cell-free in vitro assay competed with TBL4K and
L-852,646 for binding to the SPP active site. This finding is further
evidence that PS1 and SPP are of the same type of aspartic protease
(30, 44, 45).
-Secretase/PS1 Inhibitors on SPP in
Live Cells--
We next tested the inhibitory potency of
-secretase/PS1 inhibitors on SPP in a cellular assay system. Besides
cleaving signal peptides, SPP also catalyzes the processing of HCV core
protein and promotes its release from the ER membrane and trafficking to lipid droplets in the cytosol (33). When SPP is inhibited, the core
protein is not processed and remains anchored in the ER membrane by the
C-terminal hydrophobic transmembrane region. We therefore could
investigate SPP activity in tissue culture cells expressing HCV
proteins and monitor the processing of core protein either by detecting
core protein by Western blot analysis (Fig.
4A), or by visualizing its
intracellular localization using indirect immunofluorescence (Fig. 4,
B and C).
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Fig. 4.
Effect of
-secretase/PS1 inhibitors on SPP activity in live
cells. A, Western blot analysis of core protein
processing. Hepatitis C virus (HCV) core-E1-E2 polyprotein was
expressed in baby hamster kidney cells in the presence of protease
inhibitors at the indicated concentrations.
C/191, core protein processed by signal
peptidase; C/179, mature core protein processed
by signal peptidase and SPP. B, analysis of cells by
immunofluorescence. HCV core-E1-E2 was expressed in baby hamster kidney
cells and probed with a core-specific antibody and staining of lipid
droplets. C, immunofluorescence of core protein and staining
of lipid droplets with cells expressing HCV core-E1-E2 in the presence
of inhibitors.
-secretase/PS1 inhibitors
L-685,458, LY411575, and NVP-AHW700-NX efficiently inhibit SPP in the
detergent-solubilized state as well as in living cells. These compounds
prevent intramembrane proteolysis of SPP substrates, which, in turn,
cannot be released from the ER membrane, and fulfill associated
functions in the cell (46).
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-secretase/PS1 are not necessarily specific and can affect the related intramembrane-cleaving aspartic protease SPP. This finding has implications for the therapeutic strategy in the treatment of AD. To date, the therapeutic potential of
small compound inhibitors of
-secretase/PS1 was scored mainly against the possible side effects that could be expected by the concomitant inhibition on the Notch-1 signaling pathway (28). This was
evaluated by measuring the inhibition of fetal T cell maturation in the
presence of
-secretase/PS1 inhibitors (47-49). However, the results
presented in this study suggest that some of the most potent
-secretase/PS1 inhibitors can also block SPP. At first glance, our
data are discouraging in respect to developing
-secretase/PS1
inhibitors as therapeutics, because SPP plays a key role in the
processing of distinct signal peptides (30), which can have
post-targeting functions such as that of reporting proper biosynthesis
of antigen-presenting major histocompatibility class I molecules to the
immune system (32, 50). Our study, however, also identified compounds
that are more selective against either
-secretase/PS1 or SPP,
indicating that specific inhibitors may be designed but need to be
tested against the individual intramembrane-cleaving aspartic proteases.
-secretase complex has been
probed intensively, but it still remains somewhat controversial. Biotinylated photoaffinity labels, based on aspartic protease transition-state analogues that mimic the
-secretase cleavage site
in
-APP/C99, can be covalently cross-linked to PS1 (19, 20).
Furthermore,
-secretase activity is abolished by mutations of two
critical aspartate residues (ASP-257 and
ASP-385) located in the predicted transmembrane domains of
PS1 (21, 22). Although such findings support the hypothesis that PS1 is
the catalytic component of the complex, this notion was hampered by the
fact that PS1 did not share any sequence homology with other known aspartic proteases. A limited relationship to the bacterial type IV
prepilin peptidase, as revealed by Haass and co-workers (44), and the
discovery of SPP, an intramembrane-cleaving aspartic protease with
active site motifs identical to the putative ones in PS1 (29),
overruled this objection and provided further evidence that PS1 is a protease.
-secretase/PS1 and SPP. Furthermore, the active site-directed
affinity probe L-852,646, previously applied to label PS1 in
solubilized total cell membranes (19), selectively labeled SPP when
applied on detergent-solubilized ER membrane proteins. The latter also
contained PS (not shown) but only in the unprocessed form, which cannot
be labeled by L-852,646 (19). In fact, all of the effective inhibitors
competed with labeling of SPP by the transition state analogue
L-852,646 and the photoaffinity label TBL4K, which mimics
the gem-diol intermediate upon hydration in the active site. These
results suggest that the compounds investigated in this study target
the active site of SPP, and it is likely that they similarly interact
with PS1.
-secretase/PS1 and SPP, the other tested inhibitors affected both
proteases to a variable degree. Thus despite overlapping inhibitor
activities, the two proteases clearly differ in the way they interact
with the inhibitors. The small number of compounds investigated,
however, does not allow us make predictions about the specificity of a
particular compound. Modifications on a lead compound may not only
significantly increase its inhibitory potency but also can influence
compound selectivity, as shown for DAPT and its second-generation
derivative, LY411575. The new derivative is indeed ~400 times more
potent against
-secretase/PS1, but it also became an efficient
inhibitor of SPP. The potency of LY411575 against SPP, however, was
less than against
-secretase/PS1. Similarly, the transition state
analogue L-685,458 was less potent against SPP, whereas the related
compound NVP-AHW700-NX was equally effective against SPP and
-secretase/PS1. Thus, SPP and
-secretase/PS1 interact differently
with various compounds, but to determine what makes an inhibitor
selective against one or the other protease will be a major challenge
for future drug design.
-secretase/PS1 are both of pharmaceutical interest.
SPP is essential for the processing of the HCV core protein (33), and
-secretase/PS1 is implicated in the cause of AD (51). Drugs against either protease may be useful for the treatment of HCV infection or AD, but they should discriminate between the two proteases
in order to minimize side effects. An added complication, however, is that the human genome encodes four additional
homologues of SPP (29, 52, 53). It is likely that these candidate
aspartic proteases catalyze intramembrane proteolysis of so far
unidentified substrate proteins. In analogy to known
intramembrane-cleaving proteases, they may promote the release of
bioactive peptides and proteins such as signaling molecules and
transcription factors (30). Because all of these proteins contain
motifs identical to the active site motifs of SPP and
-secretase/PS1, compounds like the ones tested in the present study
may well target the SPP-like candidate proteases too. Therefore,
compound specificity will be even more important. In the future, the
development of effective therapeutic agents targeting
-secretase/PS1
or SPP will challenge the chemists and may require systematic probing of all human intramembrane-cleaving aspartic proteases.
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ACKNOWLEDGEMENTS |
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We thank J. McLauchlan for antibodies JM122
and R308, F. Checler for compound JKL2, and R. Ortmann and U. Neumann
for development of the A ELISA methodology.
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FOOTNOTES |
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* This work was supported by grants from the Center of Neuroscience Zurich, the National Competence Center for Research "Neuronal Plasticity and Repair," and the Swiss National Science Foundation (to B. M.) and by a Boehringer Ingelheim fellowship (to M. K. L.).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.
These authors contributed equally to this work.
¶ To whom correspondence should be addressed. Tel.: 41-1-632-6347; Fax: 41-1-632-1269; E-mail: bruno.martoglio@bc.biol.ethz.ch.
Published, JBC Papers in Press, March 5, 2003, DOI 10.1074/jbc.M301372200
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ABBREVIATIONS |
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The abbreviations used are:
AD, Alzheimer's disease;
-APP,
-amyloid precursor protein;
C99, residual membrane-anchored stub of 99 residues;
ER, endoplasmic
reticulum;
HCV, hepatitis C virus;
HEK, human embryonic kidney;
HLA, human lymphocyte antigen;
PS, presenilin;
SPP, signal peptide
peptidase;
Bicine, N,N-bis(2-hydroxyethyl)glycine;
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid;
CHAPSO, 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonic
acid;
DAPT, N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine
t-butyl ester..
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