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

2003; Elsevier BV; Volume: 278; Issue: 19 Linguagem: Inglês

10.1074/jbc.m301372200

ISSN

1083-351X

Autores

Andreas Weihofen, Marius K. Lemberg, Elena Friedmann, Heinrich Rueeger, Albert Schmitz, Paolo Paganetti, Giorgio Rovelli, Bruno Martoglio,

Tópico(s)

Biochemical and Structural Characterization

Resumo

Presenilin is implicated in the pathogenesis of Alzheimer's disease. It is thought to constitute the catalytic subunit of the γ-secretase complex that catalyzes intramembrane cleavage of औ-amyloid precursor protein, the last step in the generation of amyloidogenic Aऔ peptides. The latter are major constituents of amyloid plaques in the brain of Alzheimer's disease patients. Inhibitors of γ-secretase are considered potential therapeutics for the treatment of this disease because they prevent production of Aऔ 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 γ-secretase inhibitors directed against presenilin may also inhibit signal peptide peptidase. Here we demonstrate that some of the most potent known γ-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 Aऔ generation without affecting other intramembrane-cleaving aspartic proteases. Presenilin is implicated in the pathogenesis of Alzheimer's disease. It is thought to constitute the catalytic subunit of the γ-secretase complex that catalyzes intramembrane cleavage of औ-amyloid precursor protein, the last step in the generation of amyloidogenic Aऔ peptides. The latter are major constituents of amyloid plaques in the brain of Alzheimer's disease patients. Inhibitors of γ-secretase are considered potential therapeutics for the treatment of this disease because they prevent production of Aऔ 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 γ-secretase inhibitors directed against presenilin may also inhibit signal peptide peptidase. Here we demonstrate that some of the most potent known γ-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 Aऔ generation without affecting other intramembrane-cleaving aspartic proteases. Alzheimer's disease औ-amyloid precursor protein residual membrane-anchored stub of 99 residues endoplasmic reticulum hepatitis C virus human embryonic kidney human lymphocyte antigen presenilin signal peptide peptidase N,N-bis(2-hydroxyethyl)glycine 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonic acid N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycinet-butyl ester. 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 Aऔ peptides Aऔ40 and Aऔ42, respectively (1Glenner G.G. Wong C.W. Biochem. Biophys. Res. Commun. 1984; 120: 885-890Crossref PubMed Scopus (4244) Google Scholar). The amyloid cascade hypothesis casually links the generation of amyloid plaques with the neuropathological changes accompanying the symptoms typical of this disease (2Selkoe D.J. Physiol. Rev. 2001; 81: 741-766Crossref PubMed Scopus (5196) Google Scholar). Aऔ peptides are generated from the type I transmembrane protein औ-APP (औ-amyloid precursor protein) by sequential proteolysis (3Kang J. Lemaire H.G. Unterbeck A. Salbaum J.M. Masters C.L. Grzeschik K.H. Multhaup G. Beyreuther K. Muller-Hill B. Nature. 1987; 325: 733-736Crossref PubMed Scopus (3957) Google Scholar). The protein is first cleaved in the exoplasmic domain by the औ-site APP-cleaving enzyme (BACE) to release the ectodomain (4Vassar R. Bennett B.D. Babu-Khan S. Kahn S. Mendiaz E.A. Denis P. Teplow D.B. Ross S. Amarante P. Loeloff R. Luo Y. Fisher S. Fuller J. Edenson S. Lile J. Jarosinski M.A. Biere A.L. Curran E. Burgess T. Louis J.C. Collins F. Treanor J. Rogers G. Citron M. Science. 1999; 286: 735-741Crossref PubMed Scopus (3327) Google Scholar, 5Sinha S. Anderson J.P. Barbour R. Basi G.S. Caccavello R. Davis D. Doan M. Dovey H.F. Frigon N. Hong J. Jacobson-Croak K. Jewett N. Keim P. Knops J. Lieberburg I. et al.Nature. 1999; 402: 537-540Crossref PubMed Scopus (1487) Google Scholar). The residual membrane-anchored stub of 99 residues (C99) is subsequently cleaved in the center of the transmembrane region by γ-secretase (6Haass C. Steiner H. Trends Cell Biol. 2002; 12: 556-562Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). The resulting cleavage products, an Aऔ peptide and the amyloid intracellular domain (AICD), are liberated from the lipid bilayer toward the exoplasm and cytosol, respectively (7Cao X. Sudhof T.C. Science. 2001; 293: 115-120Crossref PubMed Scopus (1058) Google Scholar, 8Kimberly W.T. Zheng J.B. Guenette S.Y. Selkoe D.J. J. Biol. Chem. 2001; 276: 40288-40292Abstract Full Text Full Text PDF PubMed Scopus (397) Google Scholar, 9Leissring M.A. Murphy M.P. Mead T.R. Akbari Y. Sugarman M.C. Jannatipour M. Anliker B. Muller U. Saftig P. De Strooper B. Wolfe M.S. Golde T.E. LaFerla F.M. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 4697-4702Crossref PubMed Scopus (254) Google Scholar). To date, the majority of characterized familial AD mutations are clustered along the presenilin-1 (PS1) gene (10Sherrington R. Rogaev E.I. Liang Y. Rogaeva E.A. Levesque G. Ikeda M. Chi H. Lin C. Li G. Holman K. Tsuda T. Mar L. Foncin J.-F. Bruni A.C. Montesi M.P. et al.Nature. 1995; 375: 754-760Crossref PubMed Scopus (3599) Google Scholar, 11Levy-Lahad E. Wasco W. Poorkaj P. Romano D.M. Oshima J. Pettingell W.H., Yu, C.E. Jondro P.D. Schmidt S.D. Wang K. et al.Science. 1995; 269: 973-977Crossref PubMed Scopus (2241) Google Scholar). They are thought to accelerate disease onset by increasing the Aऔ42/Aऔ40 ratio (12Selkoe D.J. Nature. 1999; 399 (6738 Suppl.): A23-A31Crossref PubMed Scopus (1534) Google Scholar). 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 Aऔ (13Jarrett J.T. Berger E.P. Lansbury Jr., P.T. Biochemistry. 1993; 32: 4693-4697Crossref PubMed Scopus (1768) Google Scholar). It has been shown that PS1 plays a key role in transport and maturation of औ-APP (14Kaether C. Lammich S. Edbauer D. Ertl M. Rietdorf J. Capell A. Steiner H. Haass C. J. Cell Biol. 2002; 158: 551-561Crossref PubMed Scopus (169) Google Scholar). It is also an essential component of the γ-secretase complex (6Haass C. Steiner H. Trends Cell Biol. 2002; 12: 556-562Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar), and several lines of evidences suggest that PS1 may constitute the catalytic subunit of this multi-subunit protease (15Wolfe M.S. J. Med. Chem. 2001; 44: 2039-2060Crossref PubMed Scopus (141) Google Scholar). For example, several aspartic protease transition state analogues have been found to inhibit γ-secretase activity and target PS1 (16Wolfe M.S. Citron M. Diehl T.S. Xia W. Donkor I.O. Selkoe D.J. J. Med. Chem. 1998; 41: 6-9Crossref PubMed Scopus (219) Google Scholar, 17Wolfe M.S. Xia W. Moore C.L. Leatherwood D.D. Ostaszewski B. Rahmati T. Donkor I.O. Selkoe D.J. Biochemistry. 1999; 38: 4720-4727Crossref PubMed Scopus (309) Google Scholar, 18Shearman M.S. Beher D. Clarke E.E. Lewis H.D. Harrison T. Hunt P. Nadin A. Smith A.L. Stevenson G. Castro J.L. Biochemistry. 2000; 39: 8698-8704Crossref PubMed Scopus (368) Google Scholar, 19Li Y.M. Xu M. Lai M.T. Huang Q. Castro J.L. DiMuzio-Mower J. Harrison T. Lellis C. Nadin A. Neduvelil J.G. Register R.B. Sardana M.K. Shearman M.S. Smith A.L. Shi X.P. Yin K.C. Shafer J.A. Gardell S.J. Nature. 2000; 405: 689-694Crossref PubMed Scopus (867) Google Scholar, 20Esler W.P. Kimberly W.T. Ostaszewski B.L. Diehl T.S. Moore C.L. Tsai J.Y. Rahmati T. Xia W. Selkoe D.J. Wolfe M.S. Nat. Cell Biol. 2000; 2: 428-434Crossref PubMed Scopus (508) Google Scholar), and conservative mutations of putative active site aspartates in PS1 result in the loss of γ-secretase activity (21Wolfe M.S. Xia W. Ostaszewski B.L. Diehl T.S. Kimberly W.T. Selkoe D.J. Nature. 1999; 398: 513-517Crossref PubMed Scopus (1699) Google Scholar, 22Steiner H. Romig H. Pesold B. Philipp U. Baader M. Citron M. Loetscher H. Jacobsen H. Haass C. Biochemistry. 1999; 38: 14600-14605Crossref PubMed Scopus (87) Google Scholar). Thus, in recent years, the development of small molecular weight compounds aimed at reducing γ-secretase/PS1 activity as a possible therapeutic strategy for AD has attracted major attention. Several potent inhibitors that affect γ-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 (23Josien H. Curr. Opin. Drug Discov. Dev. 2002; 5: 513-525PubMed Google Scholar). The major concern related to this approach is that γ-secretase/PS1 not only catalyzes the processing of C99, but it is also required for the processing of other transmembrane proteins such as CD44 (24Lammich S. Okochi M. Takeda M. Kaether C. Capell A. Zimmer A.-K. Edbauer D. Walter J. Steiner H. Haass C. J. Biol. Chem. 2002; 277: 44754-44759Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar), the tyrosine kinase receptor Erb4 (25Ni C.Y. Murphy M.P. Golde T.E. Carpenter G. Science. 2001; 294: 2179-2181Crossref PubMed Scopus (759) Google Scholar, 26Lee H.J. Jung K.M. Huang Y.Z. Bennett L.B. Lee J.S. Mei L. Kim T.W. J. Biol. Chem. 2002; 277: 6318-6323Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar), and the Notch receptor family (27De Strooper B. Annaert W. Cupers P. Saftig P. Craessaerts K. Mumm J.S. Schroeter E.H. Schrijvers V. Wolfe M.S. Ray W.J. Goate A. Kopan R. Nature. 1999; 398: 518-522Crossref PubMed Scopus (1808) Google Scholar, 28Mumm J.S. Kopan R. Dev. Biol. 2000; 228: 151-165Crossref PubMed Scopus (850) Google Scholar). Recently, we identified the intramembrane-cleaving protease SPP (for signal peptide peptidase) that contains motifs YD and LGLGD characteristic for GXGD aspartic proteases (29Weihofen A. Binns K. Lemberg M.K. Ashman K. Martoglio B. Science. 2002; 296: 2215-2218Crossref PubMed Scopus (457) Google Scholar). 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 γ-secretase/PS1 complex (6Haass C. Steiner H. Trends Cell Biol. 2002; 12: 556-562Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 30Weihofen A. Martoglio B. Trends Cell Biol. 2003; 13: 71-78Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar). 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 (29Weihofen A. Binns K. Lemberg M.K. Ashman K. Martoglio B. Science. 2002; 296: 2215-2218Crossref PubMed Scopus (457) Google Scholar, 31Lemberg M.K. Martoglio B. Mol. Cell. 2002; 10: 735-744Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar). 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 (32Lemberg M.K. Bland F.A. Weihofen A. Braud V.M. Martoglio B. J. Immunol. 2001; 167: 6441-6446Crossref PubMed Scopus (153) Google Scholar). 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 (33McLauchlan J. Lemberg M.K. Hope G. Martoglio B. EMBO J. 2002; 21: 3980-3988Crossref PubMed Scopus (397) Google Scholar). 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 γ-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 γ-secretase/PS1 inhibitors on SPP activity. We first tested the compounds for their potency in blocking Aऔ generation in intact cells as well as inhibiting solubilized γ-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. L-658,485 (34Nadin A. Sanchez J.M. Neduvelil J.G. Thomas S.R. Tetrahedron. 2001; 57: 1861-1864Crossref Scopus (36) Google Scholar), L-852,646 (19Li Y.M. Xu M. Lai M.T. Huang Q. Castro J.L. DiMuzio-Mower J. Harrison T. Lellis C. Nadin A. Neduvelil J.G. Register R.B. Sardana M.K. Shearman M.S. Smith A.L. Shi X.P. Yin K.C. Shafer J.A. Gardell S.J. Nature. 2000; 405: 689-694Crossref PubMed Scopus (867) Google Scholar), DAPT (WO 9822494), LY411575 (WO 9828268), (Z-LL)2-ketone (35Weihofen A. Lemberg M.K. Ploegh H.L. Bogyo M. Martoglio B. J. Biol. Chem. 2000; 275: 30951-30956Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar), and TBL4K (29Weihofen A. Binns K. Lemberg M.K. Ashman K. Martoglio B. Science. 2002; 296: 2215-2218Crossref PubMed Scopus (457) Google Scholar) were synthesized as described previously. NVP-AHW700-NX was synthesized according to methods reported for a related compound (36Beher D. Wrigley J.D. Nadin A. Evin G. Masters C.L. Harrison T. Castro J.L. Shearman M.S. J. Biol. Chem. 2001; 276: 45394-45402Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). The purity of each compound was checked by1H 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 (37Petit A. Bihel F. Alves da Costa C. Pourquie O. Checler F. Kraus J.L. Nat. Cell Biol. 2001; 3: 507-511Crossref PubMed Scopus (193) Google Scholar) was kindly provided by F. Checler and pepstatin A was purchased from Sigma. Inhibition of γ-secretase activity in live cells was assayed by quantifying the generation of secreted Aऔ. In brief, human embryonic kidney cells (HEK)-293 cells stably transfected with औ-APP carrying the Swedish mutation (38Mullan M. Crawford F. Axelman K. Houlden H. Lilius L. Winblad B. Lannfelt L. Nat. Genet. 1992; 1: 345-347Crossref PubMed Scopus (1198) Google Scholar,39Schrader-Fischer G. Paganetti P.A. Brain Res. 1996; 716: 91-100Crossref PubMed Scopus (67) Google Scholar) 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 ॖl of conditioned medium were removed for determination of Aऔ levels by sandwich enzyme-linked immunosorbent assay using the Aऔ40-specific monoclonal antibody 25H10 raised against the free C-terminal peptide, MVGGVV, of Aऔ40. The monoclonal औ1antibody (39Schrader-Fischer G. Paganetti P.A. Brain Res. 1996; 716: 91-100Crossref PubMed Scopus (67) Google Scholar) 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 Aऔ40 peptide (Bachem) were run in parallel. For testing γ-secretase in vitro, detergent-solubilized γ-secretase activity was prepared from HEK-293 cells (40Li Y.M. Lai M.T. Xu M. Huang Q. DiMuzio-Mower J. Sardana M.K. Shi X.P. Yin K.C. Shafer J.A. Gardell S.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6138-6143Crossref PubMed Scopus (501) Google Scholar) and incubated with substrate Met-C99, which was synthesized by in vitro translation (see below), and either Me2SO (27) or inhibitor at the indicated concentration. After incubation, samples were subjected to immunoprecipitation with antibody 25H10 and analyzed by SDS-PAGE and phosphorimaging using 157 polyacrylamide Tris-Bicine-urea acrylamide gels (41Wiltfang J. Smirnov A. Schnierstein B. Kelemen G. Matthies U. Klafki H.W. Staufenbiel M. Huther G. Ruther E. Kornhuber J. Electrophoresis. 1997; 18: 527-532Crossref PubMed Scopus (122) Google Scholar) and a STORM PhosphorImager (Amersham Biosciences). Reference peptide Met-Aऔ40 was synthesized byin vitro translation. γ-Secretase inhibitors were tested on SPP in a previously established in vitroassay (35Weihofen A. Lemberg M.K. Ploegh H.L. Bogyo M. Martoglio B. J. Biol. Chem. 2000; 275: 30951-30956Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). In brief, 2 ॖl of cell-free translation mixture containing [35S]methionine-labeled peptidep-PrlPP29/30 (31Lemberg M.K. Martoglio B. Mol. Cell. 2002; 10: 735-744Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar) were diluted with 35 ॖl of SPP buffer (25 mm HEPES-KOH, pH 7.6, 100 mmKOAc, 2 mm Mg(OAc)2, 1 mmdithiothreitol) and supplemented with 1 ॖl of 100× concentrated inhibitor in Me2SO. Reactions were initiated by the addition of 2 ॖl 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 157 polyacrylamide Tris-Bicine-urea acrylamide gels (41Wiltfang J. Smirnov A. Schnierstein B. Kelemen G. Matthies U. Klafki H.W. Staufenbiel M. Huther G. Ruther E. Kornhuber J. Electrophoresis. 1997; 18: 527-532Crossref PubMed Scopus (122) Google Scholar) 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 (29Weihofen A. Binns K. Lemberg M.K. Ashman K. Martoglio B. Science. 2002; 296: 2215-2218Crossref PubMed Scopus (457) Google Scholar). 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) (29Weihofen A. Binns K. Lemberg M.K. Ashman K. Martoglio B. Science. 2002; 296: 2215-2218Crossref PubMed Scopus (457) Google Scholar). Samples were analyzed by SDS-PAGE on 127 polyacrylamide Tris-glycine gels (42Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar), and biotinylated proteins were visualized by enhanced chemiluminescence (Amersham Biosciences) after Western blotting with a polyclonal anti-biotin antibody (Bethyl) (29Weihofen A. Binns K. Lemberg M.K. Ashman K. Martoglio B. Science. 2002; 296: 2215-2218Crossref PubMed Scopus (457) Google Scholar). Hepatitis C virus structural proteins C, E1, and E2 were transiently expressed in baby hamster kidney C13 cells as described previously (33McLauchlan J. Lemberg M.K. Hope G. Martoglio B. EMBO J. 2002; 21: 3980-3988Crossref PubMed Scopus (397) Google Scholar). Following electroporation with in vitro transcribed mRNA encoding the CE1E2 polyprotein, cells were diluted in growth medium at a concentration of ∼106cells/ml. An 0.25-ml cell suspension was diluted with 0.25 ml of growth medium containing either 27 Me2SO, or 27 100× 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 (43Hope R.G. McLauchlan J. J. Gen. Virol. 2000; 81: 1913-1925Crossref PubMed Scopus (192) Google Scholar) (gift from J. McLauchlan) and staining of lipid droplets (43Hope R.G. McLauchlan J. J. Gen. Virol. 2000; 81: 1913-1925Crossref PubMed Scopus (192) Google Scholar). For Western blot analysis, proteins were first separated by SDS-PAGE using 137 polyacrylamide Tris-glycine gels, transferred to polyvinylidene difluoride membranes, and probed with polyclonal core-specific antibody R308 (43Hope R.G. McLauchlan J. J. Gen. Virol. 2000; 81: 1913-1925Crossref PubMed Scopus (192) Google Scholar) (gift from J. McLauchlan). Bound antibody was detected by enhanced chemiluminescence. The potency of γ-secretase/PS1 inhibitors L-685,458 (18Shearman M.S. Beher D. Clarke E.E. Lewis H.D. Harrison T. Hunt P. Nadin A. Smith A.L. Stevenson G. Castro J.L. Biochemistry. 2000; 39: 8698-8704Crossref PubMed Scopus (368) Google Scholar), L-852,646 (19Li Y.M. Xu M. Lai M.T. Huang Q. Castro J.L. DiMuzio-Mower J. Harrison T. Lellis C. Nadin A. Neduvelil J.G. Register R.B. Sardana M.K. Shearman M.S. Smith A.L. Shi X.P. Yin K.C. Shafer J.A. Gardell S.J. Nature. 2000; 405: 689-694Crossref PubMed Scopus (867) Google Scholar), 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 (35Weihofen A. Lemberg M.K. Ploegh H.L. Bogyo M. Martoglio B. J. Biol. Chem. 2000; 275: 30951-30956Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar) and TBL4K (29Weihofen A. Binns K. Lemberg M.K. Ashman K. Martoglio B. Science. 2002; 296: 2215-2218Crossref PubMed Scopus (457) Google Scholar) 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 γ-secretase/PS1 inhibitors and affect generation of Aऔ peptides and compared the potency of the two compounds with known γ-secretase/PS1 inhibitors DAPT, L-685,458, and LY411575 (Fig. 1B). Stably transfected HEK-293 cells expressing औ-APP were treated with various concentrations of inhibitor. Following incubation for 24 h, medium was removed and analyzed for Aऔ peptides in a sandwich enzyme-linked immunosorbent assay. Compounds LY411575 and NVP-AHW700-NX efficiently inhibited Aऔ generation with IC50 values of 0.4 nm and 0.62 ॖm, respectively, as well as the previously described inhibitors L-685,458 (0.46 ॖm) and DAPT (0.17 ॖm). In contrast, (Z-LL)2-ketone did not inhibit the generation of soluble Aऔ40 up to a concentration of 100 ॖm. The latter finding was confirmed in a cell-free in vitroassay using detergent-solubilized HEK cell membranes containing γ-secretase/PS1 activity (Fig. 1C). As a substrate, we used Met-C99, which was synthesized by cell-free in vitrotranslation. This peptide corresponded to the natural substrate of γ-secretase/PS1, C99, with an additional N-terminal methionine required to initiate peptide synthesis. After incubation, samples were subjected to immunoprecipitation with an Aऔ40-specific antiserum and analyzed by SDS-PAGE and phosphorimaging. As expected, the γ-secretase/PS1 inhibitor DAPT (5 ॖm) blocked the generation of Aऔ40. The SPP inhibitor (Z-LL)2-ketone, in contrast, did not affect production of Aऔ40 up to a concentration of 100 ॖm. We next investigated the effect of γ-secretase/PS1 inhibitors on SPP activity, first in a previously described cell-free in vitro assay (35Weihofen A. Lemberg M.K. Ploegh H.L. Bogyo M. Martoglio B. J. Biol. Chem. 2000; 275: 30951-30956Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). A radiolabeled SPP substrate, peptide p-PrlPP29/30 (31Lemberg M.K. Martoglio B. Mol. Cell. 2002; 10: 735-744Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar), 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-longp-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 γ-secretase/PS1 inhibitors L-685,458, L-852,646, LY411575, and NVP-AHW700-NX efficiently inhibited cleavage ofp-PrlPP29 with apparent IC50 values ranging from 8 to ∼100 nm. Interestingly, the γ-secretase/PS1 inhibitor DAPT, which is a less potent derivative of LY411575, had no effect on SPP activity at concentrations up to 100 ॖm (Fig. 2). Also, pepstatin A and JKL2, both of which were reported to inhibit γ-secretase/PS1 activity (37Petit A. Bihel F. Alves da Costa C. Pourquie O. Checler F. Kraus J.L. Nat. Cell Biol. 2001; 3: 507-511Crossref PubMed Scopus (193) Google Scholar, 40Li Y.M. Lai M.T. Xu M. Huang Q. DiMuzio-Mower J. Sardana M.K. Shi X.P. Yin K.C. Shafer J.A. Gardell S.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6138-6143Crossref PubMed Scopus (501) Google Scholar), did not affect SPP at concentrations up to 100 ॖm. To test whether the effective γ-secretase/PS1 inhibitors affect SPP by binding to the active site of SPP, we labeled the protease with the previously described photoaffinity label, TBL4K (29Weihofen A. Binns K. Lemberg M.K. Ashman K. Martoglio B. Science. 2002; 296: 2215-2218Crossref PubMed Scopus (457) Google Scholar) 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 γ-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. To further demonstrate that some of the γ-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 (19Li Y.M. Xu M. Lai M.T. Huang Q. Castro J.L. DiMuzio-Mower J. Harrison T. Lellis C. Nadin A. Neduvelil J.G. Register R.B. Sardana M.K. Shearman M.S. Smith A.L. Shi X.P. Yin K.C. Shafer J.A. Gardell S.J. Nature. 2000; 405: 689-694Crossref PubMed Scopus (867) Google Scholar). 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 (30Weihofen A. Martoglio B. Trends Cell Biol. 2003; 13: 71-78Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 44Steiner H. Kostka M. Romig H. Basset G. Pesold B. Hardy J. Capell A. Meyn L. Grim M.L. Baumeister R. Fechteler K. Haass C. Nat. Cell Biol. 2000; 2: 848-851Crossref PubMed Scopus (255) Google Scholar, 45Wolfe M.S. Selkoe D.J. Science. 2002; 296: 2156-2157Crossref PubMed Scopus (56) Google Scholar). We next tested the inhibitory potency of γ-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 (33McLauchlan J. Lemberg M.K. Hope G. Martoglio B. EMBO J. 2002; 21: 3980-3988Crossref PubMed Scopus (397) Google Scholar). 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). 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 ॖm (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 ॖm, 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 γ-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 (46Hegde R.S. Mol. Cell. 2002; 10: 697-698Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). In the present study we have demonstrated that aspartic protease inhibitors directed against γ-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 γ-secretase/PS1 was scored mainly against the possible side effects that could be expected by the concomitant inhibition on the Notch-1 signaling pathway (28Mumm J.S. Kopan R. Dev. Biol. 2000; 228: 151-165Crossref PubMed Scopus (850) Google Scholar). This was evaluated by measuring the inhibition of fetal T cell maturation in the presence of γ-secretase/PS1 inhibitors (47Hadland B.K. Manley N.R. Su D. Longmore G.D. Moore C.L. Wolfe M.S. Schroeter E.H. Kopan R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7487-7491Crossref PubMed Scopus (190) Google Scholar, 48Doerfler P. Shearman M.S. Perlmutter R.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9312-9317Crossref PubMed Scopus (155) Google Scholar, 49Radtke F. Wilson A. Ernst B. MacDonald H.R. Immunol. Rev. 2002; 187: 65-74Crossref PubMed Scopus (67) Google Scholar). However, the results presented in this study suggest that some of the most potent γ-secretase/PS1 inhibitors can also block SPP. At first glance, our data are discouraging in respect to developing γ-secretase/PS1 inhibitors as therapeutics, because SPP plays a key role in the processing of distinct signal peptides (30Weihofen A. Martoglio B. Trends Cell Biol. 2003; 13: 71-78Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar), 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 (32Lemberg M.K. Bland F.A. Weihofen A. Braud V.M. Martoglio B. J. Immunol. 2001; 167: 6441-6446Crossref PubMed Scopus (153) Google Scholar, 50Braud V.M. Allan D.S. O'Callaghan C.A. Soderstrom K. D'Andrea A. Ogg G.S. Lazetic S. Young N.T. Bell J.I. Phillips J.H. Lanier L.L. McMichael A.J. Nature. 1998; 391: 795-799Crossref PubMed Scopus (1770) Google Scholar). Our study, however, also identified compounds that are more selective against either γ-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 γ-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 γ-secretase cleavage site in औ-APP/C99, can be covalently cross-linked to PS1 (19Li Y.M. Xu M. Lai M.T. Huang Q. Castro J.L. DiMuzio-Mower J. Harrison T. Lellis C. Nadin A. Neduvelil J.G. Register R.B. Sardana M.K. Shearman M.S. Smith A.L. Shi X.P. Yin K.C. Shafer J.A. Gardell S.J. Nature. 2000; 405: 689-694Crossref PubMed Scopus (867) Google Scholar, 20Esler W.P. Kimberly W.T. Ostaszewski B.L. Diehl T.S. Moore C.L. Tsai J.Y. Rahmati T. Xia W. Selkoe D.J. Wolfe M.S. Nat. Cell Biol. 2000; 2: 428-434Crossref PubMed Scopus (508) Google Scholar). Furthermore, γ-secretase activity is abolished by mutations of two critical aspartate residues (asp-257 andasp-385) located in the predicted transmembrane domains of PS1 (21Wolfe M.S. Xia W. Ostaszewski B.L. Diehl T.S. Kimberly W.T. Selkoe D.J. Nature. 1999; 398: 513-517Crossref PubMed Scopus (1699) Google Scholar, 22Steiner H. Romig H. Pesold B. Philipp U. Baader M. Citron M. Loetscher H. Jacobsen H. Haass C. Biochemistry. 1999; 38: 14600-14605Crossref PubMed Scopus (87) Google Scholar). 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 (44Steiner H. Kostka M. Romig H. Basset G. Pesold B. Hardy J. Capell A. Meyn L. Grim M.L. Baumeister R. Fechteler K. Haass C. Nat. Cell Biol. 2000; 2: 848-851Crossref PubMed Scopus (255) Google Scholar), and the discovery of SPP, an intramembrane-cleaving aspartic protease with active site motifs identical to the putative ones in PS1 (29Weihofen A. Binns K. Lemberg M.K. Ashman K. Martoglio B. Science. 2002; 296: 2215-2218Crossref PubMed Scopus (457) Google Scholar), 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 γ-secretase/PS1 and SPP. Furthermore, the active site-directed affinity probe L-852,646, previously applied to label PS1 in solubilized total cell membranes (19Li Y.M. Xu M. Lai M.T. Huang Q. Castro J.L. DiMuzio-Mower J. Harrison T. Lellis C. Nadin A. Neduvelil J.G. Register R.B. Sardana M.K. Shearman M.S. Smith A.L. Shi X.P. Yin K.C. Shafer J.A. Gardell S.J. Nature. 2000; 405: 689-694Crossref PubMed Scopus (867) Google Scholar), 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 (19Li Y.M. Xu M. Lai M.T. Huang Q. Castro J.L. DiMuzio-Mower J. Harrison T. Lellis C. Nadin A. Neduvelil J.G. Register R.B. Sardana M.K. Shearman M.S. Smith A.L. Shi X.P. Yin K.C. Shafer J.A. Gardell S.J. Nature. 2000; 405: 689-694Crossref PubMed Scopus (867) Google Scholar). 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 γ-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 γ-secretase/PS1, but it also became an efficient inhibitor of SPP. The potency of LY411575 against SPP, however, was less than against γ-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 γ-secretase/PS1. Thus, SPP and γ-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 γ-secretase/PS1 are both of pharmaceutical interest. SPP is essential for the processing of the HCV core protein (33McLauchlan J. Lemberg M.K. Hope G. Martoglio B. EMBO J. 2002; 21: 3980-3988Crossref PubMed Scopus (397) Google Scholar), and γ-secretase/PS1 is implicated in the cause of AD (51Wolfe M.S. Nat. Rev. Drug Discov. 2002; 1: 859-866Crossref PubMed Scopus (180) Google Scholar). 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 (29Weihofen A. Binns K. Lemberg M.K. Ashman K. Martoglio B. Science. 2002; 296: 2215-2218Crossref PubMed Scopus (457) Google Scholar, 52Grigorenko A.P. Moliaka Y.K. Korovaitseva G.I. Rogaev E.I. Biochemistry (Mosc.). 2002; 67: 826-835Crossref PubMed Scopus (56) Google Scholar, 53Ponting C.P. Hutton M. Nyborg A. Baker M. Jansen K. Golde T.E. Hum. Mol. Genet. 2002; 11: 1037-1044Crossref PubMed Scopus (154) Google Scholar). 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 (30Weihofen A. Martoglio B. Trends Cell Biol. 2003; 13: 71-78Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar). Because all of these proteins contain motifs identical to the active site motifs of SPP and γ-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 γ-secretase/PS1 or SPP will challenge the chemists and may require systematic probing of all human intramembrane-cleaving aspartic proteases. We thank J. McLauchlan for antibodies JM122 and R308, F. Checler for compound JKL2, and R. Ortmann and U. Neumann for development of the Aऔ ELISA methodology.

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