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• W2132989968 abstract "γ-Secretase is an unusual protease with an intramembrane catalytic site that cleaves many type I membrane proteins, including the amyloid β-protein (Aβ) precursor (APP) and the Notch receptor. Genetic and biochemical studies have identified four membrane proteins as components of γ-secretase: heterodimeric presenilin composed of its N- and C-terminal fragments, nicastrin, Aph-1, and Pen-2. Here we demonstrated that certain compounds, including protein kinase inhibitors and their derivatives, act directly on purified γ-secretase to selectively block cleavage of APP- but not Notch-based substrates. Moreover, ATP activated the generation of the APP intracellular domain and Aβ, but not the generation of the Notch intracellular domain by the purified protease complex, and was a direct competitor of the APP-selective inhibitors, as were other nucleotides. In accord, purified γ-secretase bound specifically to an ATP-linked resin. Finally, a photoactivable ATP analog specifically labeled presenilin 1-C-terminal fragments in purified γ-secretase preparations; the labeling was blocked by ATP itself and APP-selective γ-secretase inhibitors. We concluded that a nucleotide-binding site exists within γ-secretase, and certain compounds that bind to this site can specifically modulate the generation of Aβ while sparing Notch. Drugs targeting the γ-secretase nucleotide-binding site represent an attractive strategy for safely treating Alzheimer disease. γ-Secretase is an unusual protease with an intramembrane catalytic site that cleaves many type I membrane proteins, including the amyloid β-protein (Aβ) precursor (APP) and the Notch receptor. Genetic and biochemical studies have identified four membrane proteins as components of γ-secretase: heterodimeric presenilin composed of its N- and C-terminal fragments, nicastrin, Aph-1, and Pen-2. Here we demonstrated that certain compounds, including protein kinase inhibitors and their derivatives, act directly on purified γ-secretase to selectively block cleavage of APP- but not Notch-based substrates. Moreover, ATP activated the generation of the APP intracellular domain and Aβ, but not the generation of the Notch intracellular domain by the purified protease complex, and was a direct competitor of the APP-selective inhibitors, as were other nucleotides. In accord, purified γ-secretase bound specifically to an ATP-linked resin. Finally, a photoactivable ATP analog specifically labeled presenilin 1-C-terminal fragments in purified γ-secretase preparations; the labeling was blocked by ATP itself and APP-selective γ-secretase inhibitors. We concluded that a nucleotide-binding site exists within γ-secretase, and certain compounds that bind to this site can specifically modulate the generation of Aβ while sparing Notch. Drugs targeting the γ-secretase nucleotide-binding site represent an attractive strategy for safely treating Alzheimer disease. Alzheimer disease is characterized by the progressive accumulation of amyloid β-protein (Aβ) 3The abbreviations used are: Aβamyloid β-proteinADAlzheimer diseaseAPPamyloid β-protein precursorAICDAPP intracellular domainCHOChinese hamster ovaryCTFC-terminal fragmentELISAenzyme-linked immunosorbent assayGSTglutathione S-transferaseNCTnicastrinNTFN-terminal fragmentPCPhosphatidylcholinePEPhosphatidylethanolaminePSpresenilinCHAPSO3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonic acidATPγSadenosine 5′-O-(thiotriphosphate)BNblue nativeHPLChigh pressure liquid chromatographyMALDI-TOFmatrix-assisted laser desorption ionization time-of-flight. in brain regions subserving memory and cognition (1Selkoe D.J. Physiol. Rev. 2001; 81: 741-766Crossref PubMed Scopus (5196) Google Scholar). Sequential proteolytic cleavages of the amyloid β-protein precursor (APP) by the β- and γ-secretases generate the amyloid β-protein (Aβ) (1Selkoe D.J. Physiol. Rev. 2001; 81: 741-766Crossref PubMed Scopus (5196) Google Scholar). β-Secretase is a single membrane-spanning aspartyl protease expressed at high levels in neurons (2Vassar R. Citron M. Neuron. 2000; 27: 419-422Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar). γ-Secretase is also an aspartyl protease but with an unprecedented intramembranous catalytic site (3Wolfe 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, 4Esler W.P. Kimberly W.T. Ostaszewski B.L. Ye W. Diehl T.S. Selkoe D.J. Wolfe M.S. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 2720-2725Crossref PubMed Scopus (345) Google Scholar) that is required for the cleavage of a wide range of type I membrane proteins that include APP and the Notch receptors (for a review see Ref. 5Kopan R. Ilagan M.X. Nat. Rev. Mol. Cell Biol. 2004; 5: 499-504Crossref PubMed Scopus (499) Google Scholar). We recently reported a specific and reproducible procedure for the high grade purification of active human γ-secretase and characterized various factors that affect its activity in vitro (6Fraering P.C. Ye W. Strub J.M. Dolios G. LaVoie M.J. Ostaszewski B.L. Van Dorsselaer A. Wang R. Selkoe D.J. Wolfe M.S. Biochemistry. 2004; 43: 9774-9789Crossref PubMed Scopus (203) Google Scholar). In further investigating the properties of the purified enzyme, we have observed that ATP can activate purified γ-secretase in vitro by up to 2-fold. This observation is in agreement with the recent report of Netzer et al. (7Netzer W.J. Dou F. Cai D. Veach D. Jean S. Li Y. Bornmann W.G. Clarkson B. Xu H. Greengard P. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 12444-12449Crossref PubMed Scopus (170) Google Scholar) that γ-secretase-mediated generation of Aβ in a mouse N2a neuroblastoma cell-free system is ATP-dependent. These authors also found that imatinib mesylate (Gleevec, formerly STI571), a selective Abl kinase inhibitor approved to treat chronic myelogenous leukemia (8Druker B.J. Tamura S. Buchdunger E. Ohno S. Segal G.M. Fanning S. Zimmermann J. Lydon N.B. Nat. Med. 1996; 2: 561-566Crossref PubMed Scopus (3167) Google Scholar, 9Druker B.J. Lydon N.B. J. Clin. Investig. 2000; 105: 3-7Crossref PubMed Scopus (812) Google Scholar, 10Mauro M.J. O'Dwyer M. Heinrich M.C. Druker B.J. J. Clin. Oncol. 2002; 20: 325-334Crossref PubMed Scopus (199) Google Scholar), inhibited γ-secretase cleavage of APP without affecting Notch processing in an N2a cell-free system, in intact N2a cells expressing human APP, and in rat primary neurons (7Netzer W.J. Dou F. Cai D. Veach D. Jean S. Li Y. Bornmann W.G. Clarkson B. Xu H. Greengard P. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 12444-12449Crossref PubMed Scopus (170) Google Scholar). Another compound with a pyrido-(2,3-d)pyrimidine structure (called inhibitor 2 (7Netzer W.J. Dou F. Cai D. Veach D. Jean S. Li Y. Bornmann W.G. Clarkson B. Xu H. Greengard P. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 12444-12449Crossref PubMed Scopus (170) Google Scholar) or PD173955 (11Moasser M.M. Srethapakdi M. Sachar K.S. Kraker A.J. Rosen N. Cancer Res. 1999; 59: 6145-6152PubMed Google Scholar)) showed a similar effect (7Netzer W.J. Dou F. Cai D. Veach D. Jean S. Li Y. Bornmann W.G. Clarkson B. Xu H. Greengard P. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 12444-12449Crossref PubMed Scopus (170) Google Scholar). Both Gleevec and inhibitor 2 are known to inhibit Abl kinase by targeting its ATP-binding site (12Wisniewski D. Lambek C.L. Liu C. Strife A. Veach D.R. Nagar B. Young M.A. Schindler T. Bornmann W.G. Bertino J.R. Kuriyan J. Clarkson B. Cancer Res. 2002; 62: 4244-4255PubMed Google Scholar, 13Nagar B. Bornmann W.G. Pellicena P. Schindler T. Veach D.R. Miller W. Clarkson B. Kuriyan J. Cancer Res. 2002; 62: 4236-4243PubMed Google Scholar, 14Nagar B. Hantschel O. Young M. Scheffzek K. Veach D. Bornmann W. Clarkson B. Superti-Furga G. Kuriyan J. Cell. 2003; 112: 859-871Abstract Full Text Full Text PDF PubMed Scopus (690) Google Scholar, 15Strife A. Wisniewski D. Liu C. Lambek C.L. Darzynkiewicz Z. Silver R.T. Clarkson B. Mol. Cancer Res. 2003; 1: 176-185PubMed Google Scholar, 16Hantschel O. Nagar B. Guettler S. Kretzschmar J. Dorey K. Kuriyan J. Superti-Furga G. Cell. 2003; 112: 845-857Abstract Full Text Full Text PDF PubMed Scopus (360) Google Scholar), but cells deficient in this enzyme were still sensitive to Gleevec with respect to lowering Aβ production (7Netzer W.J. Dou F. Cai D. Veach D. Jean S. Li Y. Bornmann W.G. Clarkson B. Xu H. Greengard P. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 12444-12449Crossref PubMed Scopus (170) Google Scholar). The mechanism by which these two compounds affect γ-secretase cleavage of APP is unknown. Because Gleevec and inhibitor 2 target several protein-tyrosine kinases besides Abl, Netzer et al. (7Netzer W.J. Dou F. Cai D. Veach D. Jean S. Li Y. Bornmann W.G. Clarkson B. Xu H. Greengard P. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 12444-12449Crossref PubMed Scopus (170) Google Scholar) suggested that platelet-derived growth factor receptor, Src kinase, or c-kit might be involved. Another proposed mechanism involves an effect on the localization of γ-secretase or APP in a way that prevents interaction of enzyme with substrate. amyloid β-protein Alzheimer disease amyloid β-protein precursor APP intracellular domain Chinese hamster ovary C-terminal fragment enzyme-linked immunosorbent assay glutathione S-transferase nicastrin N-terminal fragment Phosphatidylcholine Phosphatidylethanolamine presenilin 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonic acid adenosine 5′-O-(thiotriphosphate) blue native high pressure liquid chromatography matrix-assisted laser desorption ionization time-of-flight. A central concern about inhibiting γ-secretase to lower Aβ production in AD is that this should also interfere with Notch processing and lead to severe toxicity because of interference with cell differentiation. Indeed, significant adverse effects of γ-secretase inhibitors caused by blocking Notch signaling have been described in preclinical animal studies (17Wong G.T. Manfra D. Poulet F.M. Zhang Q. Josien H. Bara T. Engstrom L. Pinzon-Ortiz M. Fine J.S. Lee H.J. Zhang L. Higgins G.A. Parker E.M. J. Biol. Chem. 2004; 279: 12876-12882Abstract Full Text Full Text PDF PubMed Scopus (658) Google Scholar, 18Harrison T. Churcher I. Beher D. Curr. Opin. Drug Discovery Dev. 2004; 7: 709-719PubMed Google Scholar, 19Searfoss G.H. Jordan W.H. Calligaro D.O. Galbreath E.J. Schirtzinger L.M. Berridge B.R. Gao H. Higgins M.A. May P.C. Ryan T.P. J. Biol. Chem. 2003; 278: 46107-46116Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar, 20Milano J. McKay J. Dagenais C. Foster-Brown L. Pognan F. Gadient R. Jacobs R.T. Zacco A. Greenberg B. Ciaccio P.J. Toxicol. Sci. 2004; 82: 341-358Crossref PubMed Scopus (477) Google Scholar). Because Netzer et al. (7Netzer W.J. Dou F. Cai D. Veach D. Jean S. Li Y. Bornmann W.G. Clarkson B. Xu H. Greengard P. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 12444-12449Crossref PubMed Scopus (170) Google Scholar) showed that Gleevec and inhibitor 2 inhibited APP but not Notch cleavage in their systems, we investigated the effects of selected protein-tyrosine kinase inhibitors on Aβ production and on Notch substrate cleavage using isolated, purified γ-secretase. Cell Lines and Cultures—HeLa S3 cells, the Chinese hamster ovary (CHO) γ-30 cell line (co-expressing human PS1, FLAG-Pen-2, and Aph1α2-HA), and the S-1 CHO cell line (co-expressing human PS1, FLAG-Pen-2, Aph1α2-HA, and NCT-GST) were cultured as described previously (6Fraering P.C. Ye W. Strub J.M. Dolios G. LaVoie M.J. Ostaszewski B.L. Van Dorsselaer A. Wang R. Selkoe D.J. Wolfe M.S. Biochemistry. 2004; 43: 9774-9789Crossref PubMed Scopus (203) Google Scholar, 21Kimberly W.T. Esler W.P. Ye W. Ostaszewski B.L. Gao J. Diehl T. Selkoe D.J. Wolfe M.S. Biochemistry. 2003; 42: 137-144Crossref PubMed Scopus (106) Google Scholar, 22Fraering P.C. LaVoie M.J. Ye W. Ostaszewski B.L. Kimberly W.T. Selkoe D.J. Wolfe M.S. Biochemistry. 2004; 43: 323-333Crossref PubMed Scopus (119) Google Scholar). Purification of γ-Secretase and in Vitro γ-Secretase Assays—The multistep procedure for the high grade purification of human γ-secretase from the S-1 cells was performed as described previously (6Fraering P.C. Ye W. Strub J.M. Dolios G. LaVoie M.J. Ostaszewski B.L. Van Dorsselaer A. Wang R. Selkoe D.J. Wolfe M.S. Biochemistry. 2004; 43: 9774-9789Crossref PubMed Scopus (203) Google Scholar). In vitro γ-secretase assays using the recombinant APP-based substrate C100FLAG and the recombinant Notch-based substrate N100FLAG were performed as reported previously (4Esler W.P. Kimberly W.T. Ostaszewski B.L. Ye W. Diehl T.S. Selkoe D.J. Wolfe M.S. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 2720-2725Crossref PubMed Scopus (345) Google Scholar, 21Kimberly W.T. Esler W.P. Ye W. Ostaszewski B.L. Gao J. Diehl T. Selkoe D.J. Wolfe M.S. Biochemistry. 2003; 42: 137-144Crossref PubMed Scopus (106) Google Scholar). Basically, the proteolytic reaction mixtures contained C100FLAG and N100FLAG substrate at a concentration of 1 μm, purified γ-secretase solubilized in 0.2% CHAPSO/HEPES, pH 7.5, at 10-fold dilution from stock (stock = the M2 anti-FLAG-eluted fraction in the purification protocol from S-1 cells (6Fraering P.C. Ye W. Strub J.M. Dolios G. LaVoie M.J. Ostaszewski B.L. Van Dorsselaer A. Wang R. Selkoe D.J. Wolfe M.S. Biochemistry. 2004; 43: 9774-9789Crossref PubMed Scopus (203) Google Scholar)), 0.025% phosphatidylethanolamine (PE), and 0.10% phosphatidylcholine (PC). All the reactions were stopped by adding 0.5% SDS, and the samples were assayed for Aβ40 and Aβ42 by ELISA as described (23Xia W. Zhang J. Ostaszewski B.L. Kimberly W.T. Seubert P. Koo E.H. Shen J. Selkoe D.J. Biochemistry. 1998; 37: 16465-16471Crossref PubMed Scopus (172) Google Scholar). The capture antibodies were 2G3 (to Aβ residues 33–40) for the Aβ40 species and 21F12 (to Aβ residues 33–42) for the Aβ42 species. Inhibitors—Powder containing Gleevec (Novartis) was dissolved from capsules or tablets in a mixture composed of ethyl acetate and aqueous saturated sodium bicarbonate solution. The organic layer was washed several times with brine, dried on sodium sulfate, and evaporated under vacuum. Gleevec was purified and analyzed by reverse phase-HPLC using a Vydac C18 preparative column (10 μm, 2.2 × 25 cm) and a C18 analytical column (5 μm, 0.46 × 25 cm), respectively. Chromatographic separations were performed at a flow of 1.5 ml/min, with a gradient of 0–100% MeOH in water over 30 min. This isolated material is referred to as “Gleevec extract.” Purified Gleevec was from Sequoia Research Products, UK. Final purity and characterization of the two Gleevec extracts (from capsules and tablets, respectively) and the purified Gleevec (Sequoia Research Products, UK) were performed by MALDI-TOF mass spectroscopy (Applied Biosystems Voyager System 4036). Gleevec was detected with a m/z of (M + H)+ = 494 g/mol (expected m/z of (M + H)+ = 493 g/mol). The well characterized γ-secretase inhibitor III-31C was prepared as described previously (4Esler W.P. Kimberly W.T. Ostaszewski B.L. Ye W. Diehl T.S. Selkoe D.J. Wolfe M.S. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 2720-2725Crossref PubMed Scopus (345) Google Scholar). Bryostatin-1 was purchased from Biomol. All compounds listed in Fig. 4 were purchased from TOCRIS-UK; the structure and action of these compounds are described in TABLE ONE (modified from TOCRIS-UK in the Supplemental Material). All inhibitors were added to the reaction mixtures from a Me2SO stock (final Me2SO concentration of 1%, which alone did not affect γ-secretase activity). Western Blotting and Antibodies—For Western analysis of PS1-NTF, PS1-CTF, Aph1α2-HA, FLAG-Pen-2, and NCT-GST, the samples were run on 4–20% Tris-glycine polyacrylamide gels, transferred to polyvinylidene difluoride, and probed with Ab14 (for PS1-NTF, 1:2000; a gift of S. Gandy), 13A11 (for PS1-CTF, 5 μg/ml; a gift of Elan Pharmaceuticals), 3F10 (for Aph1α2-HA, 50 ng/ml; Roche Applied Science), anti-FLAG M2 (for FLAG-Pen-2, 1:1000; Sigma), or αGST antibodies (for NCT-GST, 1:3000; Sigma). Samples from the γ-secretase activity assays (above) were run on 4–20% Tris-glycine gels and transferred to polyvinylidene difluoride membranes to detect AICD-FLAG with anti-FLAG M2 antibodies (1:1000, Sigma) and NICD-FLAG with Notch Ab1744 antibody (1:1000, Cell Signaling Technology), which is selective for the N terminus of NICD; the same samples were transferred to nitrocellulose membranes to detect Aβ with the anti-Aβ 6E10 antibody. Levels of AICD-FLAG and NICD-FLAG were estimated by densitometry using AlphaEase/Spot Denso (Alpha Innotech Corp.). Purified γ-Secretase and Binding to ATP-immobilized Resins—The purified γ-secretase was diluted 10-fold from stock (6Fraering P.C. Ye W. Strub J.M. Dolios G. LaVoie M.J. Ostaszewski B.L. Van Dorsselaer A. Wang R. Selkoe D.J. Wolfe M.S. Biochemistry. 2004; 43: 9774-9789Crossref PubMed Scopus (203) Google Scholar) in 50 mm HEPES buffer, pH 7.0, containing 0.2 or 1% CHAPSO, 150 mm NaCl, 5 mm MgCl2, 5 mm CaCl2 and incubated overnight, in the presence or absence of 50 mm ATP (Sigma), with ATP-agarose (ATP attached to agarose through the ribose hydroxyls, Sigma catalog number A-4793) or ATP-acrylamide (ATP attached to acrylamide through the γ-phosphate; Novagen catalog number 71438-3). Each resin was washed three times with 0.2 or 1% CHAPSO/HEPES buffer, and the bound proteins were collected in 2× Laemmli sample buffer, resolved on 4–20% Tris-glycine gels, and transferred to polyvinylidene difluoride membranes to detect NCT-GST, PS1-NTF, Aph1-HA, PS1-CTF, and FLAG-Pen2 as described above. Photoaffinity Labeling Experiments—8-Azido-[γ-32P]ATP (18 Ci/mmol) was purchased from Affinity Labeling Technology (Lexington, KY). For the photoaffinity labeling of the purified γ-secretase, the enzyme was diluted 10-fold from stock (6Fraering P.C. Ye W. Strub J.M. Dolios G. LaVoie M.J. Ostaszewski B.L. Van Dorsselaer A. Wang R. Selkoe D.J. Wolfe M.S. Biochemistry. 2004; 43: 9774-9789Crossref PubMed Scopus (203) Google Scholar) in 50 mm HEPES buffer, pH 7.0, containing 0.2% CHAPSO, 150 mm NaCl, 5 mm MgCl2, 5 mm CaCl2, 0.025% PE, and 0.10% PC. The samples were exposed to UV light for 5 min (hand-held UV lamp at 254 nm; UVP model UVGL-25) on ice, and the reaction was quenched with 1 mm dithiothreitol. The proteins were diluted in 0.5% CHAPSO/HEPES buffer and incubated overnight for affinity precipitation with GSH resin as described previously (6Fraering P.C. Ye W. Strub J.M. Dolios G. LaVoie M.J. Ostaszewski B.L. Van Dorsselaer A. Wang R. Selkoe D.J. Wolfe M.S. Biochemistry. 2004; 43: 9774-9789Crossref PubMed Scopus (203) Google Scholar, 22Fraering P.C. LaVoie M.J. Ye W. Ostaszewski B.L. Kimberly W.T. Selkoe D.J. Wolfe M.S. Biochemistry. 2004; 43: 323-333Crossref PubMed Scopus (119) Google Scholar). The unbound nucleotides were removed by washing the resin three times and then the washed proteins were resuspended in Laemmli sample buffer. For the photoaffinity labeling of the purified γ-secretase followed by the BN-PAGE analysis, the enzyme was diluted in 0.1% digitonin/TBS, exposed to UV light for 5 min, and directly loaded onto a 5–13.5% BN-polyacrylamide gel. For the photoaffinity labeling of endogenous γ-secretase, HeLa S3 membranes (the equivalent of 3.0 × 108 cells) were incubated with 22.5 μm 8-azido-[γ-32P]ATP (10 μCi per reaction), 50 mm HEPES, pH 7.0, 150 mm NaCl, 5 mm MgCl2, and 5 mm CaCl2 in a total volume of 60 μl for 10 min at 37 °C. The resuspended membranes were exposed to UV light as described above. The unbound nucleotides were removed by washing the membranes three times and then the washed membranes were resuspended for 1 h in 0.5 ml of 1% CHAPSO/HEPES, pH 7.4. The solubilized proteins were diluted 1:2 in HEPES buffer (final CHAPSO concentration = 0.5%) and incubated overnight with X81 antibody for immunoprecipitation, as described previously (6Fraering P.C. Ye W. Strub J.M. Dolios G. LaVoie M.J. Ostaszewski B.L. Van Dorsselaer A. Wang R. Selkoe D.J. Wolfe M.S. Biochemistry. 2004; 43: 9774-9789Crossref PubMed Scopus (203) Google Scholar, 22Fraering P.C. LaVoie M.J. Ye W. Ostaszewski B.L. Kimberly W.T. Selkoe D.J. Wolfe M.S. Biochemistry. 2004; 43: 323-333Crossref PubMed Scopus (119) Google Scholar). Samples were electrophoresed on 4–20% Tris-glycine gels and autoradiographed (BioMax MS films used with BioMax Transcreen HE (Eastman Kodak Co.)). ATPase Assays—[α-32P]ATP (11.9 Ci/mmol) was purchased from Affinity Labeling Technology (Lexington, KY). The purified γ-secretase was prepared as described for the photoaffinity labeling experiments; 5 μCi of [α-32P]ATP was added; the reactions were incubated at 37 °C, and at the indicated time points aliquots were removed and reactions stopped by addition of 10% SDS. A total of 2 μl of each stopped reaction was analyzed by TLC on polyethyleneimine cellulose plastic sheets (Baker-Flex, Germany) with 0.75 m KH2PO4, pH 3.5, as the running buffer to separate ATP from ADP. To identify hydrolysis products, a reaction of [α-32P]ATP incubated in the presence of 0.005 units of canine kidney phosphatase (Sigma) was loaded. Samples were autoradiographed as described above. Nonhydrolyzed ATP Can Activate the Generation of AICD and Aβ but Not the Generation of NICD by Purified γ-Secretase—We took advantage of our highly purified γ-secretase complexes to investigate factors that might affect the cleavage of APP, using a C100FLAG substrate consisting of the β-CTF (C99) portion of APP (amino acids 596–695) plus a Met at the N terminus and a FLAG tag at the C terminus (4Esler W.P. Kimberly W.T. Ostaszewski B.L. Ye W. Diehl T.S. Selkoe D.J. Wolfe M.S. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 2720-2725Crossref PubMed Scopus (345) Google Scholar, 24Li 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). We observed that increased concentrations of ATP promoted a dose-dependent rise in the production of both AICD-FLAG (Fig. 1A, compare lanes 2–6 with lane 1) and Aβ (both Aβ40 and Aβ42), reaching an ∼1.75-fold increase at 1–5 mm as estimated by Aβ ELISA (Fig. 1B). Densitometry of the AICD-FLAG bands (Fig. 1A) showed that the fold increases were similar to those seen for Aβ by ELISA in Fig. 1B. These findings are in good agreement with the reported data of Netzer et al. (7Netzer W.J. Dou F. Cai D. Veach D. Jean S. Li Y. Bornmann W.G. Clarkson B. Xu H. Greengard P. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 12444-12449Crossref PubMed Scopus (170) Google Scholar), which showed that the addition of 1–3 mm ATP in a cell-free membrane preparation (derived from mouse N2a cells expressing human APP-695) results in an ∼2-fold increase in Aβ production. Next, we addressed whether ATP can stimulate the cleavage of a Notch-based substrate (N100FLAG) in a similar manner and under the same conditions as for C100FLAG. N100FLAG is an analogous Notch-based substrate corresponding to residues Val-1711 to Glu-1809 from the mouse Notch-1 receptor (plus a Met and a FLAG tag at the N and C termini, respectively, just like C100) (21Kimberly W.T. Esler W.P. Ye W. Ostaszewski B.L. Gao J. Diehl T. Selkoe D.J. Wolfe M.S. Biochemistry. 2003; 42: 137-144Crossref PubMed Scopus (106) Google Scholar). We found that increased concentrations of ATP did not alter the generation of NICD-FLAG (Fig. 1A, compare lanes 2–6 with lane 1), and this was confirmed by densitometry. ATP stores energy in the form of a chemical bond and releases it in the process of hydrolysis, providing a readily available energy supply for many enzymatic reactions. Thus, we intended to determine whether the peptidase activity of γ-secretase is associated with ATP hydrolysis. As shown in Fig. 1C, the purified γ-secretase incubated in the presence (lanes 21–25) or absence (lanes 6–10) of C100FLAG substrate did not increase the hydrolysis of [α-32P]ATP into [α-32P]ADP when compared with the hydrolysis occurring in the reaction buffer alone (lanes 11–15). This result indicates that ATP hydrolysis is not required for the peptidase activity of γ-secretase. We further found that 1 mm levels of the nonhydrolyzable ATP analog, ATPγS, also resulted in an ∼1.35 increase in the generation of Aβ40 and Aβ42 from C100FLAG substrate by purified γ-secretase (data not shown), supporting our observation that ATP hydrolysis is not required for the effect. Gleevec Itself Is Not a Direct γ-Secretase Inhibitor; However, a Gleevec Extract Inhibits the Generation of Aβ by Purified γ-Secretase Without Affecting the Cleavage of a Notch-based Recombinant Substrate—Because ATP activated the purified γ-secretase complex (Fig. 1, A and B), we used this preparation to examine the effects of a Gleevec extract (prepared from capsules and characterized as described in detail under “Materials and Methods”) on the cleavage efficiency of C100FLAG substrate. We first confirmed that III-31C (4Esler W.P. Kimberly W.T. Ostaszewski B.L. Ye W. Diehl T.S. Selkoe D.J. Wolfe M.S. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 2720-2725Crossref PubMed Scopus (345) Google Scholar) and DAPT (25Dovey H.F. John V. Anderson J. Chen L.Z. de Saint Andrieu P. Fang L.Y. Freedman S.B. Folmer B. Goldbach E. Holsztynska E.J. Hu K.L. Johnson-Wood K.L. Kennedy S.L. Kholodenko D. Knops J.E. Latimer L.H. Lee M. Liao Z. Lieberburg I.M. Motter R.N. Mutter L.C. Nietz J. Quinn K.P. Sacchi K.L. Seubert P.A. Shopp G.M. Thorsett E.D. Tung J.S. Wu J. Yang S. Yin C.T. Schenk D.B. May P.C. Altstiel L.D. Bender M.H. Boggs L.N. Britton T.C. Clemens J.C. Czilli D.L. Dieckman-McGinty D.K. Droste J.J. Fuson K.S. Gitter B.D. Hyslop P.A. Johnstone E.M. Li W.Y. Little S.P. Mabry T.E. Miller F.D. Audia J.E. J. Neurochem. 2001; 76: 173-181Crossref PubMed Scopus (803) Google Scholar), two well characterized inhibitors of γ-secretase, inhibited both C100FLAG and N100FLAG cleavage by our purified γ-secretase with similar potencies (III-31C, IC50 of ∼50 nm for cleavage of C100FLAG, as estimated by ELISA and densitometry, and ∼100 nm for cleavage of N100FLAG, as estimated by densitometry; DAPT, IC50 of ∼100 nm for cleavage of C100FLAG, as estimated by ELISA and densitometry, and ∼100 nm for cleavage of N100FLAG, as estimated by densitometry) (Fig. 2A, and ELISA data not shown). We then probed the effects of our Gleevec extract (extracted from capsules and isolated by HPLC as described under “Materials and Methods”) on Aβ production by the purified γ-secretase. The cleavage products, Met-Aβ40 and Met-Aβ42, (which we designate Aβ40 and Aβ42 for simplicity) were quantified by ELISA (Fig. 2B) and also detected by blotting with the 6E10 anti-Aβ antibody (Fig. 2C). The other proteolytic product, FLAG-tagged AICD, was detected with anti-FLAG M2 antibodies (Fig. 2C). Our Gleevec extract inhibited the generation of Aβ40, Aβ42, and AICD in a concentration-dependent fashion and with a similar potency, yielding an approximate IC50 value (estimated by Aβ ELISA) of ∼75 μm (Fig. 2, B and C). Next, we examined the effects of our Gleevec extract on the cleavage of N100FLAG by the purified protease. The Gleevec extract did not inhibit the generation of NICD-FLAG, even at concentrations >10-fold the IC50 value for the generation of Aβ40 and Aβ42 from C100FLAG substrate (i.e. at 1 mm) (Fig. 2C, lane 7). Similarly, the Gleevec extract was found to inhibit the generation of Aβ by endogenous γ-secretase solubilized from HeLa cell membranes without affecting the cleavage of N100FLAG substrate (Fig. 2D). Because pH is an important factor modulating the activity of the purified γ-secretase (6Fraering P.C. Ye W. Strub J.M. Dolios G. LaVoie M.J. Ostaszewski B.L. Van Dorsselaer A. Wang R. Selkoe D.J. Wolfe M.S. Biochemistry. 2004; 43: 9774-9789Crossref PubMed Scopus (203) Google Scholar), the pH of all the above reactions was checked and found to be consistently at pH 7.4, even at high concentrations (1 mm) of the Gleevec extract. Because several other compounds (impurities or degradation products probably generated during the extraction and purification procedures, m/z of 200.1, 247.6, 268.2, 277.1, 286.6, 308.9, 332.9, 350.0, 380.1, 395.1, and 516.2) were detected in the Gleevec preparation (Fig. 2E, left panel and data not shown), we decided to examine the effect on the purified γ-secretase of two additional Gleevec samples prepared from two different sources as follows: Gleevec extracted from tablets as described in detail under “Materials and Methods” and purified Gleevec (Sequoia Research Products, UK). Final purity of the two Gleevec samples described above was addressed by MALDI-TOF mass spectroscopy (Fig. 2E, middle and right panels, respectively). We found that these two Gleevec samples did not inhibit the generation of AICD, Aβ40, and Aβ42 by the purified γ-secretase (Fig. 2E, middle and right panels, respectively, and ELISA data not shown). Because Gleevec (m/z of (M + H)+ = 494) was found in the three different Gleevec samples, our data strongly suggest that Gleevec is not the active compound found in the Gleevec preparation extracted from capsules. Most interestingly, the analyses by mass spectroscopy of the three Gleevec samples revealed several compounds (m/z of 200.1, 308.9, 332.9, 350.0, and 516.2) that were only present in the active Gleevec preparation (Fig. 2E, the compounds identified specifically in the active Gleevec preparation are labeled with arrowheads), leaving open the possibility that one or more of those compounds are active toward γ-secretase. Also, a very minor peak at 286.6 in the inactive extract is a major peak in the active extract (Fig. 2E, asterisk). Although purification and characterization of each of the compounds found in our active Gleevec extract will now be necessary for the identificatio" @default.
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• W2132989968 title "γ-Secretase Substrate Selectivity Can Be Modulated Directly via Interaction with a Nucleotide-binding Site" @default.
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