Targeting Presenilin-type Aspartic Protease Signal Peptide Peptidase with gamma -Secretase Inhibitors*

Andreas WeihofenDagger, Marius K. LembergDagger, Elena Friedmann, Heinrich Rueeger§, Albert Schmitz§, Paolo Paganetti§, Giorgio Rovelli§, and Bruno Martoglio

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

    ABSTRACT
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INTRODUCTION
EXPERIMENTAL PROCEDURES
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Presenilin is implicated in the pathogenesis of Alzheimer's disease. It is thought to constitute the catalytic subunit of the gamma -secretase complex that catalyzes intramembrane cleavage of beta -amyloid precursor protein, the last step in the generation of amyloidogenic Abeta peptides. The latter are major constituents of amyloid plaques in the brain of Alzheimer's disease patients. Inhibitors of gamma -secretase are considered potential therapeutics for the treatment of this disease because they prevent production of Abeta 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 gamma -secretase inhibitors directed against presenilin may also inhibit signal peptide peptidase. Here we demonstrate that some of the most potent known gamma -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 Abeta generation without affecting other intramembrane-cleaving aspartic proteases.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 Abeta peptides Abeta 40 and Abeta 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). Abeta peptides are generated from the type I transmembrane protein beta -APP (beta -amyloid precursor protein) by sequential proteolysis (3). The protein is first cleaved in the exoplasmic domain by the beta -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 gamma -secretase (6). The resulting cleavage products, an Abeta peptide and the amyloid intracellular domain (AICD), are liberated from the lipid bilayer toward the exoplasm and cytosol, respectively (7-9).

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 Abeta 42/Abeta 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 Abeta (13). It has been shown that PS1 plays a key role in transport and maturation of beta -APP (14). It is also an essential component of the gamma -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 gamma -secretase activity and target PS1 (16-20), and conservative mutations of putative active site aspartates in PS1 result in the loss of gamma -secretase activity (21, 22). Thus, in recent years, the development of small molecular weight compounds aimed at reducing gamma -secretase/PS1 activity as a possible therapeutic strategy for AD has attracted major attention. Several potent inhibitors that affect gamma -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 gamma -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).

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 gamma -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.

The common features of SPP and PS1 raise the question of whether gamma -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 gamma -secretase/PS1 inhibitors on SPP activity. We first tested the compounds for their potency in blocking Abeta generation in intact cells as well as inhibiting solubilized gamma -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.

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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.

gamma -Secretase Assays-- Inhibition of gamma -secretase activity in live cells was assayed by quantifying the generation of secreted Abeta . In brief, human embryonic kidney cells (HEK)-293 cells stably transfected with beta -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 Abeta levels by sandwich enzyme-linked immunosorbent assay using the Abeta 40-specific monoclonal antibody 25H10 raised against the free C-terminal peptide, MVGGVV, of Abeta 40. The monoclonal beta 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 Abeta 40 peptide (Bachem) were run in parallel.

For testing gamma -secretase in vitro, detergent-solubilized gamma -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-Abeta 40 was synthesized by in vitro translation.

SPP Assay and Affinity Labeling-- gamma -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).

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.

    RESULTS
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INTRODUCTION
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Introducing gamma -Secretase/PS1 Inhibitors and Potency of SPP Inhibitor against gamma -Secretase/PS1-- The potency of gamma -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 gamma -secretase/PS1 inhibitors and affect generation of Abeta peptides and compared the potency of the two compounds with known gamma -secretase/PS1 inhibitors DAPT, L-685,458, and LY411575 (Fig. 1B). Stably transfected HEK-293 cells expressing beta -APP were treated with various concentrations of inhibitor. Following incubation for 24 h, medium was removed and analyzed for Abeta peptides in a sandwich enzyme-linked immunosorbent assay. Compounds LY411575 and NVP-AHW700-NX efficiently inhibited Abeta 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 Abeta 40 up to a concentration of 100 µM.


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Fig. 1.   Inhibitory potency of gamma -secretase/PS1 inhibitors and (Z-LL)2-ketone. A, chemical structure of inhibitors and photoaffinity labels used in this study. B, inhibition of gamma -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 Abeta 40 secreted into the medium. C, effect of (Z-LL)2-ketone on detergent-solubilized gamma -secretase/PS1 activity. Radiolabeled gamma -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 Abeta 40-specific antibody. ref, reference peptide Met-Abeta 40.

The latter finding was confirmed in a cell-free in vitro assay using detergent-solubilized HEK cell membranes containing gamma -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 gamma -secretase/PS1, C99, with an additional N-terminal methionine required to initiate peptide synthesis. After incubation, samples were subjected to immunoprecipitation with an Abeta 40-specific antiserum and analyzed by SDS-PAGE and phosphorimaging. As expected, the gamma -secretase/PS1 inhibitor DAPT (5 µM) blocked the generation of Abeta 40. The SPP inhibitor (Z-LL)2-ketone, in contrast, did not affect production of Abeta 40 up to a concentration of 100 µM.

Inhibition of Detergent-solubilized SPP-- We next investigated the effect of gamma -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 gamma -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 gamma -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 gamma -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 gamma -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.

Active Site Labeling of SPP and Competition with gamma -Secretase/PS1 Inhibitors-- To test whether the effective gamma -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 gamma -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 gamma -secretase/PS1 inhibitors. A, labeling with TBL4K and competition. SPP was labeled with TBL4K in the presence of gamma -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.

To further demonstrate that some of the gamma -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).

Potency of gamma -Secretase/PS1 Inhibitors on SPP in Live Cells-- We next tested the inhibitory potency of gamma -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 gamma -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.

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 gamma -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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In the present study we have demonstrated that aspartic protease inhibitors directed against gamma -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 gamma -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 gamma -secretase/PS1 inhibitors (47-49). However, the results presented in this study suggest that some of the most potent gamma -secretase/PS1 inhibitors can also block SPP. At first glance, our data are discouraging in respect to developing gamma -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 gamma -secretase/PS1 or SPP, indicating that specific inhibitors may be designed but need to be tested against the individual intramembrane-cleaving aspartic proteases.

The nature of the catalytic site of the gamma -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 gamma -secretase cleavage site in beta -APP/C99, can be covalently cross-linked to PS1 (19, 20). Furthermore, gamma -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.

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 gamma -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.

Although three compounds, pepstatin A, DAPT, and (Z-LL)2-ketone, could discriminate between gamma -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 gamma -secretase/PS1, but it also became an efficient inhibitor of SPP. The potency of LY411575 against SPP, however, was less than against gamma -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 gamma -secretase/PS1. Thus, SPP and gamma -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.

SPP and gamma -secretase/PS1 are both of pharmaceutical interest. SPP is essential for the processing of the HCV core protein (33), and gamma -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 gamma -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 gamma -secretase/PS1 or SPP will challenge the chemists and may require systematic probing of all human intramembrane-cleaving aspartic proteases.

    ACKNOWLEDGEMENTS

We thank J. McLauchlan for antibodies JM122 and R308, F. Checler for compound JKL2, and R. Ortmann and U. Neumann for development of the Abeta ELISA methodology.

    FOOTNOTES

* 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.

Dagger 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

    ABBREVIATIONS

The abbreviations used are: AD, Alzheimer's disease; beta -APP, beta -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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REFERENCES

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