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Keywords:

  • Alzheimer’s disease;
  • Amyloid precursor protein;
  • Amyloid peptide;
  • Proteolysis;
  • Protease inhibitors;
  • γ-Secretase

Abstract

  1. Top of page
  2. Abstract
  3. EXPERIMENTAL PROCEDURES
  4. Synthesis of Z-VL-CHO
  5. RESULTS AND DISCUSSION
  6. Intracellular accumulation of C-terminal APP fragments on treatment with dipeptidyl aldehydes
  7. Acknowledgements
  8. References

Abstract: The carboxy-terminal ends of the 40- and 42-amino acids amyloid β-protein (Aβ) may be generated by the action of at least two different proteases termed γ(40)- and γ(42)-secretase, respectively. To examine the cleavage specificity of the two proteases, we treated amyloid precursor protein (APP)-transfected cell cultures with several dipeptidyl aldehydes including N-benzyloxycarbonyl-Leu-leucinal (Z-LL-CHO) and the newly synthesized N-benzyloxycarbonyl-Val-leucinal (Z-VL-CHO). All dipeptidyl aldehydes tested inhibited production of both Aβ1-40 and Aβ1-42. Changes in the P1 and P2 residues of these aldehydes, however, indicated that the amino acids occupying these positions are important for the efficient inhibition of γ-secretases. Peptidyl aldehydes inhibit both cysteine and serine proteases, suggesting that the two γ-secretases belong to one of these mechanistic classes. To differentiate between the two classes of proteases, we treated our cultures with the specific cysteine protease inhibitor E-64d. This agent inhibited production of secreted Aβ1-40, with a concomitant accumulation of its cellular precursor indicating that γ(40)-secretase is a cysteine protease. In contrast, this treatment increased production of secreted Aβ1-42. No inhibition of Aβ production was observed with the potent calpain inhibitor I (acetyl-Leu-Leu-norleucinal), suggesting that calpain is not involved. Together, these results indicate that γ(40)-secretase is a cysteine protease distinct from calpain, whereas γ(42)-secretase may be a serine protease. In addition, the two secretases may compete for the same substrate. Dipeptidyl aldehyde treatment of cultures transfected with APP carrying the Swedish mutation resulted in the accumulation of the β-secretase C-terminal APP fragment and a decrease of the α-secretase C-terminal APP fragment, indicating that this mutation shifts APP cleavage from the α-secretase site to the β-secretase site.

Alzheimer’s disease is a neurodegenerative disorder characterized by the presence of insoluble protein aggregates that accumulate in pathological lesions, such as neuritic amyloid plaques, cerebrovascular amyloid, and neurofibrillary tangles. The amyloid fibers of the neuritic amyloid plaque cores and cerebrovascular amyloid consist mainly of the 40-42-amino acids amyloid β-protein (Aβ), derived from the proteolytic processing of the amyloid precursor protein (APP) (for review, see Robakis, 1994). APP is a transmembrane glycoprotein processed through amyloidogenic and nonamyloidogenic pathways. In a nonamyloidogenic pathway, cleavage by α-secretase between Aβ sequence Lys16 and Leu17 of APP (Anderson et al., 1991) results in the production of an 8-kDa C-terminal APP fragment that is considered the precursor of the p3 peptides (Higaki et al., 1995; Citron et al., 1996). In the amyloidogenic pathway, proteolytic cleavage by β-secretase at the N terminus of the Aβ sequence yields a 10-kDa C-terminal fragment containing the entire Aβ sequence at its N terminus (Seubert et al., 1993). Cleavage of the 8- or 10-kDa C-terminal APP fragments by γ-secretase yields the p3 peptide or Aβ, respectively (Higaki et al., 1995; Citron et al., 1996). The C terminus of Aβ peptide is heterogeneous, ending at residue 40 or 42 (Miller et al., 1993; Roher et al., 1993), suggesting the existence of at least two different γ-secretase activities (Citron et al., 1996; Klafki et al., 1996). None of the APP secretases have been isolated and their particular class of proteases has not been identified. This is an important issue in Alzheimer’s disease, as clarification of the nature of these proteases will facilitate the design of potent Aβ inhibitors.

Recently, it was reported that certain calpain inhibitors, including several peptidyl aldehydes, inhibited γ-secretase activities (Citron et al., 1996; Klafki et al., 1996), suggesting that γ-secretase may be a calpain. The aldehyde group of these inhibitors binds to the active-site serine or cysteine of the respective protease-forming hemiacetal (or thiohemiacetal) adducts that mimic the transition state of the enzyme-substrate complexes (Thompson, 1973). To determine whether γ-secretases are cysteine or serine proteases, we used Chinese hamster ovary (CHO) cultures stably transfected with APP to examine the efficacy of the specific cysteine protease inhibitor (2S,3S)-trans-epoxysuccinyl-L-leucylamido-3-methylbutane ethyl ester (E-64d). E-64d decreased production of total Aβ in the media with a concomitant increase in production of Aβ1-42. In contrast, several dipeptidyl aldehydes, including the newly synthesized N-benzyloxycarbonyl-Val-leucinal (Z-VL-CHO) inhibited production of both Aβ isoforms. These data suggest that the activity generating Aβ1-40 involves the direct or indirect action of a cysteine protease and the activity generating Aβ1-42 includes a serine protease.

EXPERIMENTAL PROCEDURES

  1. Top of page
  2. Abstract
  3. EXPERIMENTAL PROCEDURES
  4. Synthesis of Z-VL-CHO
  5. RESULTS AND DISCUSSION
  6. Intracellular accumulation of C-terminal APP fragments on treatment with dipeptidyl aldehydes
  7. Acknowledgements
  8. References

Materials

Geneticin (G-418), minimum essential medium without phenylalanine, L-glutamine, and penicillin/streptomycin were obtained from GibcoBRL, Grand Island, NY, U.S.A.; [35S]methionine/[35S]cysteine mix was purchased from NEN, Wilmington, DE, U.S.A.; protein A-Sepharose was from Repligen, Cambridge, MA, U.S.A.; 10-20% precast Tris-tricine gels were from Novex, San Diego, CA, U.S.A. All other materials were purchased from Sigma.

The following APP antisera were used: 36395, which recognizes total Aβ and was a generous gift from Dr. S. Roberts from Bristol-Myers Squibb, Wallingford, CT, U.S.A.; 165, which recognizes Aβ1-42 specifically (see Results); R1, raised against the C-terminal 23 amino acids of APP, which recognizes full-length APP and its C-terminal fragments (Efthimiopoulos et al., 1996); 192, which was raised against the C-terminal methionine of wild-type APPsβ (Knops et al., 1995) and was a generous gift from Dr. D. Schenk, from Athena Neurosciences, San Francisco, CA, U.S.A.; and R47, which is specific for the last 16 amino acids of APPsα (652-667 amino acids of APP751) and also for the Aβ sequence 1-16 (Anderson et al., 1992).

Z-VL-CHO was synthesized in our laboratory. N-Benzyloxycarbonyl-Leu-leucinal (Z-LL-CHO) was a generous gift from Dr. S. Wilk, Department of Pharmacology, Mount Sinai School of Medicine, New York, NY, U.S.A.; N-benzyloxycarbonyl-Val-phenylalaninal (Z-VF-CHO) (Higaki et al., 1995), also known as MDL 28170, was a generous gift from Hoechst Marion Roussel, Inc., Cincinnati, OH, U.S.A.; and E-64d was purchased from Calbiochem, La Jolla, CA, U.S.A.

Synthesis of Z-VL-CHO

  1. Top of page
  2. Abstract
  3. EXPERIMENTAL PROCEDURES
  4. Synthesis of Z-VL-CHO
  5. RESULTS AND DISCUSSION
  6. Intracellular accumulation of C-terminal APP fragments on treatment with dipeptidyl aldehydes
  7. Acknowledgements
  8. References

Z-VL-CHO was synthesized from Z-Val-OH and L-leucinol as described (Levy et al., 1996). NMR, TLC, and mass spectroscopy analysis confirmed the structure and purity of the dipeptidyl aldehyde.

Cell cultures

CHO cells obtained from the American Type Tissue Culture Collection were maintained and stably transfected as previously described (Efthimiopoulos et al., 1994) with human wild-type APP751 cDNA (CHOwt) or APP751 cDNA carrying either the Swedish double mutation K651/M652 to N/L (CHOsw) or the London mutation V698G (CHOvg). Expression of the transfected APP was >20 times that of all endogenous (nontransfectant) APP signals. All peptidyl aldehydes and E-64d were used at nontoxic concentrations. Absence of toxicity was indicated by several tests including normal production of APP, MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assays (Efthimiopoulos et al., 1996), and microscopic examination of the cell cultures for signs of cell death.

Radiolabeling, drug treatment, and immunoprecipitation

CHO cells were labeled with 150 μCi/ml [35S]methionine/[35S]cysteine protein mix for 3-4 h as described (Efthimiopoulos et al., 1994). The inhibitors were dissolved in dimethyl sulfoxide and added directly to the media during labeling. Immunoprecipitations from all conditioned media (1 × 106 trichloroacetic acid-precipitable CPM) or cell extracts (1 × 107 trichloroacetic acid-precipitable CPM) were performed as previously described (Efthimiopoulos et al., 1994). The immunocomplexes were resolved on 10-20% Tris-tricine gels and visualized by autoradiography. The intensity of individual bands was quantified by image analysis as previously described (Pereira et al., 1992).

RESULTS AND DISCUSSION

  1. Top of page
  2. Abstract
  3. EXPERIMENTAL PROCEDURES
  4. Synthesis of Z-VL-CHO
  5. RESULTS AND DISCUSSION
  6. Intracellular accumulation of C-terminal APP fragments on treatment with dipeptidyl aldehydes
  7. Acknowledgements
  8. References

Effects of P1 and P2 residues of dipeptidyl aldehydes on Aβ production

The specificity and potency of peptidyl aldehyde proteinase inhibitors can be improved by modifying their amino acid sequences. In general, these sequences mimic the residues in the substrate designated as subsites P1, P2, and so on, proceeding from the scissile bond toward the N terminus of the peptide substrate. Two of the most common cleavage sites described for γ-secretase occur after the aliphatic hydrophobic sequence Val691 Val692 or I693 A694 (numbering according to APP751), yielding Aβ1-40 or Aβ1-42, respectively (Miller et al., 1993; Roher et al., 1993). A previous study showed that 200 μM of the peptidylaldehyde Z-VF-CHO significantly decreased production of total Aβ in APP-transfected CHO cells by inhibiting the activity of γ-secretase (Higaki et al., 1995). We synthesized Z-VL-CHO (see EXPERIMENTAL PROCEDURES) to examine whether substitution of a bulky aromatic residue (F) with a branched-chain aliphatic amino acid (L) at the P1 subsite of the inhibitor would modify the γ-secretase inhibitory potency of the dipeptidyl aldehyde. The effects of these inhibitors on Aβ production were examined by using metabolically labeled CHO cells stably transfected with APP751 containing either the London V698G mutation (CHOvg) or the Swedish double mutation K651/M652 to N/L (CHOsw). As shown in Fig. 1A(sw) and B(vg), a 3-h treatment of these cultures with 100 μM Z-VF-CHO led to an almost complete inhibition of total Aβ. However, treatment with 200 μM Z-VL-CHO caused only a 79% inhibition. To rule out effects not related to the aldehyde group, we tested the peptidyl alcohol Z-VL-OH, the immediate precursor of Z-VL-CHO. The latter did not affect formation of Aβ at the highest concentrations tested (results not shown). The IC50 values of Aβ inhibition were ∼45 and 100 μM for Z-VF-CHO and Z-VL-CHO, respectively (Fig. 1C), suggesting that the P1 residue is important for inhibition of Aβ formation. We also examined whether a change in the P2 residue would affect this cleavage, by substituting the Val in Z-VL-CHO with a Leu. Figure 1C shows that Z-LL-CHO was a stronger inhibitor of Aβ formation than Z-VL-CHO (IC50 35 μM vs. 100 μM, respectively). Clearly, subtle changes, like an increase of the aliphatic side chain of the amino acid at P2, have a significant effect on inhibition of Aβ production. Moreover, as the intracellular concentrations of the peptidyl aldehydes are probably lower than their media concentrations, the actual IC50 values of these inhibitors are likely lower than the values indicated here.

image

Figure 1. Effect of Z-VF-CHO, Z-LL-CHO, and Z-VL-CHO on the formation of total Aβ in CHOsw cells [A(sw)] or in CHOvg cells [B(vg)]. The antibody 36395 was used to immunoprecipitate the Aβ peptides (arrow) from the media. Lanes 1, no drug treatment; lanes 2, 100 μM Z-VF-CHO; lanes 3, 100 μM Z-LL-CHO; lanes 4, 200 μM Z-VL-CHO. Cultures were treated for 3 h during metabolic labeling. The autoradiographs shown are representative of one of at least three identical experiments for each condition tested. C: Efficacy of the inhibition of total Aβ production by Z-LL-CHO (▪), Z-VF-CHO (□), and Z-VL-CHO (○) in CHOsw cells. Activity is expressed relative to control conditions, in the absence of inhibitor. Each point represents the mean ± SD of two experiments.

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All treatments resulted in the intracellular accumulation of the 10-kDa APP fragment, which is the substrate for γ-secretase (see below), in agreement with recent evidence that peptidyl aldehydes decrease Aβ formation by inhibiting γ-secretase cleavage of the 10-kDa peptide (Higaki et al., 1995). Combined, these data suggest that a strategic approach for the design of more potent γ-secretase activity inhibitors could involve modifications/substitutions of the residues positioned at P1 and P2 of these dipeptidyl aldehydes. Further work will show whether the detected differences in the inhibitory potency of the dipeptidyl aldehydes are due to differences in their cell permeability or affinity for the active site of the affected proteases.

Media aliquots from the above cultures were analyzed further with antibody 165, which recognizes Aβ1-42 derived from the γ(42)-secretase cleavage of the 10-kDa C-terminal APP fragment (Higaki et al., 1995; Citron et al., 1996) but fails to recognize Aβ1-40. The specificity of the antibody was confirmed by the disappearance of the signal after its preadsorption with Aβ1-42 peptide (Fig. 2A, lane 2) but not with Aβ1-40 (Fig. 2A, lane 3). In addition, antibody 165 failed to recognize Aβ1-40 in western blots and ELISAs (Mehta et al., 1998). Dipeptidyl aldehydes Z-VF-CHO, Z-LL-CHO, and Z-VL-CHO reduced formation of both total Aβ and Aβ1-42.

image

Figure 2. A: Specificity of the antiserum 165 for Aβ1-42 (top arrow). The immunoprecipitates were obtained from media of untreated CHOvg cells radiolabeled with [35S]methionine/[35S]cysteine, under the following conditions: Lane 1, with antiserum 165; lane 2, with antiserum 165 preadsorbed with the peptide Aβ1-42 (10 μg/ml); lane 3, with antiserum 165 preadsorbed with the peptide Aβ1-40 (10 μg/ml). The bottom arrow indicates p317-42 that is also recognized by this antibody. B(vg) and C(sw): Effect of Z-VF-CHO (lanes 2), Z-LL-CHO (lanes 3), and Z-VL-CHO (lanes 4) on the production of Aβ1-42 in CHOvg [B(vg)] or CHOsw [C(sw)]. Antibody 165 was used to immunoprecipitate Aβ1-42 from the media. Lanes 1, no drug treatment; lanes 2, 100 μM Z-VF-CHO; lanes 3, 100 μM Z-LL-CHO; lanes 4, 200 μM Z-VL-CHO. Cultures were treated for 3 h during metabolic labeling. The positions of the molecular mass markers (kDa) are shown between the autoradiographs. The autoradiographs shown are representative of one of at least three identical experiments for each condition tested in (B) and (C).

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Peptide p317-42 is derived from APP by the sequential action of α-secretase and γ(42)-secretase. The C-terminal 8-kDa APP fragment derived by α-secretase is the immediate precursor of p317-42. Comparison of B(vg) with C(sw) in Fig. 2 shows that this peptide was readily detectable in CHOvg cultures but not in CHOsw cultures. These data are in agreement with our findings (see below) that the Swedish double mutation shifts cleavage of APP from the α-secretase site that produces the 8-kDa p3 precursor to the β-secretase site that produces the 10-kDa Aβ precursor.

Intracellular accumulation of C-terminal APP fragments on treatment with dipeptidyl aldehydes

  1. Top of page
  2. Abstract
  3. EXPERIMENTAL PROCEDURES
  4. Synthesis of Z-VL-CHO
  5. RESULTS AND DISCUSSION
  6. Intracellular accumulation of C-terminal APP fragments on treatment with dipeptidyl aldehydes
  7. Acknowledgements
  8. References

Treatment of CHOsw cultures with the dipeptidyl aldehydes resulted in the intracellular accumulation of both the 10- and the 8-kDa C-terminal APP fragments [Fig. 3A(sw), lanes 2-4] that are substrates for γ-secretases and the immediate precursors of Aβ or p3 peptides, respectively (Higaki et al., 1995; Citron et al., 1996). N-terminal radiosequencing (Ghiso et al., 1993) established that these fragments derived from APP, corresponding to cleavages by the β- and α-secretase, respectively (data not shown). Figure 3 also shows that the ratio of the accumulated 10-kDa fragment over the 8-kDa fragment was higher in CHOsw cells treated with Z-LL-CHO, compared with that in CHOvg or CHOwt cells [compare lane 3 in Fig. 3A(sw) with lanes 2 in B(vg) and C(wt)]. This finding is in accord with our data presented above [Fig. 2B(vg) and C(sw)], showing that CHOsw cells produce very little of the p317-42 peptide compared with the CHOvg cells. Together, these observations suggest that the Swedish mutation favors APP cleavage by β-secretase rather than α-secretase. As this cleavage leads to the generation of both forms of Aβ, this shift could explain the high levels of total Aβ produced from APP carrying the Swedish mutation (Citron et al., 1992; Cai et al., 1993).

image

Figure 3. Effect of Z-VF-CHO, Z-LL-CHO, and Z-VL-CHO on the accumulation of intracellular C-terminal fragments (C.T.F.) of APP751. The antiserum R1 that is specific for the last 23 amino acids (729-751) of the cytoplasmic sequence of APP751 was used to immunoprecipitate the C-terminal fragments from lysates of CHO cells. A(sw): CHO cells were transfected with APP cDNA with the Swedish double mutation. Lane 1, no drug treatment; lane 2, 100 μM Z-VF-CHO; lane 3, 100 μM Z-LL-CHO; lane 4, 200 μM Z-VL-CHO. B(vg): CHO cells were transfected with APP cDNA with the London (vg) mutation. Lane 1, control; lane 2, 100 μM Z-LL-CHO. C(wt): CHO cells were transfected with wt APP cDNA. Lane 1, control; lane 2, 100 μM Z-LL-CHO. Cultures were treated for 3 h during metabolic labeling. Arrows on the right indicate the 10 kDa (top) and 8 kDa (bottom) C.T.F. of APP751. The autoradiographs shown are representative of one of at least three identical experiments for each condition tested.

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The dipeptidyl aldehydes did not decrease the levels of radiolabeled full-length APP in the lysates of transfected CHO cells even at the highest concentrations used, indicating lack of toxicity. In addition, there was no significant decrease in the secretion of APPsα or APPsβ, the N-terminal products of the α- and β-secretase cleavage, respectively (results not shown), indicating that these agents are specific for γ-secretase, as they do not alter the activity of other secretases or the mechanism of cell secretion.

γ(40)-Secretase is a cysteine protease distinct from calpain

That γ-secretases are inhibited by dipeptidyl aldehydes suggests that these enzymes may be either serine or cysteine proteases (Thompson, 1973). To distinguish between these two mechanistic classes, we used E-64d, a cell-permeable form of the specific cysteine protease inhibitor E-64 (Tezapsidis et al., 1995). E-64d is converted to E-64 on the action of intracellular esterases. It is the only known cell-permeable agent that reacts specifically with protein-SH groups at the reactive site of cysteine proteinases. At the concentrations used (25-500 μM), E-64d is nontoxic and it does not affect serine proteases or any other class of proteases (Barrett et al., 1982; McGowan et al., 1989; Salvesen and Nagase, 1989). Accordingly, we observed no toxic effects when E-64d was added to the medium of our cultures at concentrations up to 500 μM. Treatment of our cultures with this inhibitor at concentrations ranging from 25 to 500 μM decreased total medium Aβ in a dose-dependent manner (Fig. 4A). At the highest concentration used (500 μM), E-64d inhibited production of total Aβ by ∼50% (Fig. 4B, lane 2), whereas production of the γ(42)-secretase product Aβ1-42 increased ∼3.5-fold (Fig. 4C, lane 2), suggesting that γ(42)-secretase is competing with γ(40)-secretase for the same substrate. Quantification of Aβ1-40 and Aβ1-42 by ELISAs (Mehta et al., 1998) indicated that in agreement with previous reports (Wolfe et al., 1998) total medium Aβ consisted of ∼90% Aβ1-40 and 10% Aβ1-42 (data not shown). Combined, these data suggest that 500 μM E-64d inhibited production of the γ(40)-secretase product Aβ1-40 approximately sixfold. As expected, this treatment resulted in the accumulation of the 10- and 8-kDa C-terminal fragments of APP (Fig. 4D, lane 2) but had no effect on the levels of cellular full-length APP, APPsα, or APPsβ detected with antibodies R1, R47, and 192, respectively (results not shown). Together, these data indicate that whereas γ(40)-secretase is a cysteine protease, γ(42)-secretase is not.

image

Figure 4. Effect of E-64d on the production of Aβ1-40. A: Total Aβ levels in the media are inversely correlated to the concentration of E-64d. CHOvg cells were treated in the presence of 0, 25, 50, and 500 μM E-64d. The levels of total Aβ were quantified by densitometry of the autoradiographs. The levels of Aβ detected in the culture media were calculated as percentages of those in control cells (no E-64d in the media), which were set at 100%. Data were obtained from duplicate determinations. B-D: E-64d lowers total Aβ and elevates Aβ1-42. Antibodies 36395 and 165 were used to immunoprecipitate total Aβ peptides (arrow in B) or Aβ1-42 and p317-42 (arrows in C) from the media, respectively. Cell lysates were also immunoprecipitated (see EXPERIMENTAL PROCEDURES) with the R1 antiserum, which recognizes the C-terminal fragments of APP751 (arrows in D). B-D: Lanes 1, no drug treatment; lanes 2, 500 μM E-64d. Cultures were treated for 4 h during metabolic labeling. The positions of the molecular mass markers (kDa) are shown on the left (B). The autoradiographs shown are representative of one of at least three identical experiments for each condition tested.

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Calpains are cysteine proteases and several calpain inhibitors have been shown to inhibit total Aβ production (Klafki et al., 1995, 1996; Citron et al., 1996), or in agreement with our data, to increase production of Aβ1-42 (Yamazaki et al., 1997). To further examine the possibility that γ(40)-secretase is a calpain, we used the potent calpain inhibitor acetyl-Leu-Leu-norleucinal (Ac-LLnL-CHO) (Klafki et al., 1995). Treatment of our cultures with 50 μM Ac-LLnL-CHO, which is the concentration used to inhibit calpain (Klafki et al., 1995), had no effect on Aβ production (Fig. 5A). These data suggest that neither of the two γ-secretases is a calpain, in agreement with a recent report that calpain inhibitor II [acetyl-Leu-Leu-methioninal (Ac-LLM-CHO)] also failed to inhibit Aβ production (Klafki et al., 1995).

image

Figure 5. Effect of Ac-LLnL-CHO on the production of total Aβ in CHOsw cells. The antibody 36395 was used to immunoprecipitate the Aβ peptides from the media. Lane 1, no drug treatment; lane 2, 50 μM Ac-LLnL-CHO. Cultures were treated for 6 h during metabolic labeling. The positions of the molecular mass markers (kDa) are shown on the left. The arrow indicates total Aβ.

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The design of specific inhibitors of APP secretases involved in the generation of Aβ is a strategic approach that may be of therapeutic value for treating AD. To accomplish such a task, one must consider the catalytic mechanisms and the substrate specificity of the proteases involved. Our data suggest that γ(40)-secretase is a cysteine protease. One of the characteristics of this class of proteases is the preference for hydrophobic amino acids at position P2 of peptidyl aldehyde inhibitors (Wang, 1990). Indeed, all potent γ-secretase inhibitors tested in our study, including Z-VL-CHO, Z-LL-CHO, and Z-VF-CHO, contained hydrophobic nonpolar residues in position P2 similarly to the potential scissile bond of γ-secretases (Kang et al., 1987; Roher et al., 1993). In addition, mutational analysis of the γ-secretase cleavage sites indicated that these enzymes prefer hydrophobic sequences preceding the scissile bond (Tischer and Cordell, 1996).

In summary, this study demonstrates that (1) hydrophobic dipeptidyl aldehydes inhibit both γ-secretase activities involved in the formation of Aβ; (2) γ(40)-secretase is a cysteine protease; (3) γ(42)-secretase may be a serine protease; and (4) both secretases may compete for the same substrate. This information should be useful for the design of more specific and potent inhibitors of both γ-secretases.

Acknowledgements

  1. Top of page
  2. Abstract
  3. EXPERIMENTAL PROCEDURES
  4. Synthesis of Z-VL-CHO
  5. RESULTS AND DISCUSSION
  6. Intracellular accumulation of C-terminal APP fragments on treatment with dipeptidyl aldehydes
  7. Acknowledgements
  8. References

We thank Dr. S. Wilk from the Department of Pharmacology at Mount Sinai School of Medicine, New York, NY, U.S.A. for the calpain/cathepsin B (Z-LL-CHO) inhibitor, and Hoechst Marion Russel, Inc., Cincinnati, OH, U.S.A. for the Z-VF-CHO inhibitor. We are also grateful to Dr. S. Roberts from Bristol-Myers Squibb, Wallingford, CT, U.S.A. for the 36935 antibody, and Dr. D. Schenk from Athena Neurosciences, San Francisco, CA, U.S.A. for the 192 antibody. This study was supported by National Institutes of Health grants NS-34018 (M.E.F.-P.) and AG08200 (N.K.R.), and by Alzheimer’s Association grants RG2-96-100 (N.K.R.) and RG1-96-098 (N.T.).

References

  1. Top of page
  2. Abstract
  3. EXPERIMENTAL PROCEDURES
  4. Synthesis of Z-VL-CHO
  5. RESULTS AND DISCUSSION
  6. Intracellular accumulation of C-terminal APP fragments on treatment with dipeptidyl aldehydes
  7. Acknowledgements
  8. References
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