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• W2040137618 abstract "γ-Secretase is an aspartic protease that hydrolyzes type I membrane proteins within the hydrophobic environment of the lipid bilayer. Using the CHAPSO-solubilized γ-secretase assay system, we previously found that γ-secretase activity was sensitive to the concentrations of detergent and phosphatidylcholine. This strongly suggests that the composition of the lipid bilayer has a significant impact on the activity of γ-secretase. Recently, level of secreted β-amyloid protein was reported to be attenuated by increasing levels of phosphatidylinositol 4,5-diphosphate (PI(4,5)P2) in cultured cells. However, it is not clear whether PI(4,5)P2 has a direct effect on γ-secretase activity. In this study, we found that phosphoinositides directly inhibited CHAPSO-solubilized γ-secretase activity. Interestingly, neither phosphatidylinositol nor inositol triphosphate altered γ-secretase activity. PI(4,5)P2 was also found to inhibit γ-secretase activity in CHAPSO-insoluble membrane microdomains (rafts). Kinetic analysis of β-amyloid protein production in the presence of PI(4,5)P2 suggested a competitive inhibition. Even though phosphoinositides are minor phospholipids of the membrane, the concentration of PI(4,5)P2 within the intact membrane has been reported to be in the range of 4–8 mm. The presence of PI(4,5)P2-rich rafts in the membrane has been reported in a range of cell types. Furthermore, γ-secretase is enriched in rafts. Taking these data together, we propose that phosphoinositides potentially regulate γ-secretase activity by suppressing its association with the substrate. γ-Secretase is an aspartic protease that hydrolyzes type I membrane proteins within the hydrophobic environment of the lipid bilayer. Using the CHAPSO-solubilized γ-secretase assay system, we previously found that γ-secretase activity was sensitive to the concentrations of detergent and phosphatidylcholine. This strongly suggests that the composition of the lipid bilayer has a significant impact on the activity of γ-secretase. Recently, level of secreted β-amyloid protein was reported to be attenuated by increasing levels of phosphatidylinositol 4,5-diphosphate (PI(4,5)P2) in cultured cells. However, it is not clear whether PI(4,5)P2 has a direct effect on γ-secretase activity. In this study, we found that phosphoinositides directly inhibited CHAPSO-solubilized γ-secretase activity. Interestingly, neither phosphatidylinositol nor inositol triphosphate altered γ-secretase activity. PI(4,5)P2 was also found to inhibit γ-secretase activity in CHAPSO-insoluble membrane microdomains (rafts). Kinetic analysis of β-amyloid protein production in the presence of PI(4,5)P2 suggested a competitive inhibition. Even though phosphoinositides are minor phospholipids of the membrane, the concentration of PI(4,5)P2 within the intact membrane has been reported to be in the range of 4–8 mm. The presence of PI(4,5)P2-rich rafts in the membrane has been reported in a range of cell types. Furthermore, γ-secretase is enriched in rafts. Taking these data together, we propose that phosphoinositides potentially regulate γ-secretase activity by suppressing its association with the substrate. Intramembrane proteolysis is an enigmatic event that occurs in the hydrophobic environment of the lipid bilayer. Thus far, three classes of intramembrane proteases have been identified as being involved in crucial biological processes, including cholesterol/fatty acid synthesis, epidermal growth factor receptor signaling, signal peptide processing, and type I membrane protein cleavage (1Brown M.S. Goldstein J.L. Cell. 1997; 89: 331-340Abstract Full Text Full Text PDF PubMed Scopus (2940) Google Scholar, 2Brown M.S. Ye J. Rawson R.B. Goldstein J.L. Cell. 2000; 100: 391-398Abstract Full Text Full Text PDF PubMed Scopus (1140) Google Scholar, 3Kimberly W.T. LaVoie M.J. Ostaszewski B.L. Ye W. Wolfe M.S. Selkoe D.J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 6382-6387Crossref PubMed Scopus (675) Google Scholar, 4Lee J.R. Urban S. Garvey C.F. Freeman M. Cell. 2001; 107: 161-171Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar, 5Takasugi N. Tomita T. Hayashi I. Tsuruoka M. Niimura M. Takahashi Y. Thinakaran G. Iwatsubo T. Nature. 2003; 422: 438-441Crossref PubMed Scopus (777) Google Scholar, 6Urban S. Lee J.R. Freeman M. Cell. 2001; 107: 173-182Abstract Full Text Full Text PDF PubMed Scopus (488) Google Scholar, 7Weihofen A. Binns K. Lemberg M.K. Ashman K. Martoglio B. Science. 2002; 296: 2215-2218Crossref PubMed Scopus (451) Google Scholar). γ-Secretase, an aspartyl protease, cleaves type I transmembrane proteins and is involved in the pathogenesis of Alzheimer disease (AD) 4The abbreviations used are: AD, Alzheimer disease; APP, β-amyloid precursor protein; βCTF, carboxyl-terminal fragment of APP; Aβ, β-amyloid; AICD, APP intracellular domain; sNICD-FLAG, shortened Notch intracellular domain fused with FLAG; PC, phosphatidylcholine; PI, phosphatidylinositol; PI(5)P, phosphatidylinositol 5-phosphate; PI(3,4)P2, phosphatidylinositol 3,4-diphosphate; PI(4,5)P2, phosphatidylinositol 4,5-diphosphate; PI(3,4,5)P3, phosphatidylinositol 3,4,5-triphosphate; CTF, carboxyl-terminal fragment; CHAPSO, 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonic acid; PH, pleckstrin homology; CHO, Chinese hamster ovary; PIPES, 1,4-piperazinediethanesulfonic acid; TLCK, 1-chloro-3-tosylamido-7-amino-2-heptanone or Nα-p-tosyl-l-lysine chloromethyl ketone; MES, 4-morpholineethanesulfonic acid; PLC, phospholipase C; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. 4The abbreviations used are: AD, Alzheimer disease; APP, β-amyloid precursor protein; βCTF, carboxyl-terminal fragment of APP; Aβ, β-amyloid; AICD, APP intracellular domain; sNICD-FLAG, shortened Notch intracellular domain fused with FLAG; PC, phosphatidylcholine; PI, phosphatidylinositol; PI(5)P, phosphatidylinositol 5-phosphate; PI(3,4)P2, phosphatidylinositol 3,4-diphosphate; PI(4,5)P2, phosphatidylinositol 4,5-diphosphate; PI(3,4,5)P3, phosphatidylinositol 3,4,5-triphosphate; CTF, carboxyl-terminal fragment; CHAPSO, 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonic acid; PH, pleckstrin homology; CHO, Chinese hamster ovary; PIPES, 1,4-piperazinediethanesulfonic acid; TLCK, 1-chloro-3-tosylamido-7-amino-2-heptanone or Nα-p-tosyl-l-lysine chloromethyl ketone; MES, 4-morpholineethanesulfonic acid; PLC, phospholipase C; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. (8Selkoe D.J. Physiol. Rev. 2001; 81: 741-766Crossref PubMed Scopus (5121) Google Scholar). The luminal portion of β-amyloid precursor protein (APP) is removed by β-secretase leaving the carboxyl-terminal fragment (βCTF or C99), which is the immediate substrate of γ-secretase, in the membrane (9Vassar 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 (3255) Google Scholar). γ-Secretase cleaves C99 in the middle of its transmembrane domain (γ-cleavage). This leads to release of β-amyloid (Aβ) protein that is the major component of senile plaques, one of the neuropathological hallmarks of AD (8Selkoe D.J. Physiol. Rev. 2001; 81: 741-766Crossref PubMed Scopus (5121) Google Scholar). Besides γ-cleavage, we and other groups have identified ϵ-cleavage that occurs close to the membrane/cytoplasmic boundary of APP (10Gu Y. Misonou H. Sato T. Dohmae N. Takio K. Ihara Y. J. Biol. Chem. 2001; 276: 35235-35238Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar, 11Sastre M. Steiner H. Fuchs K. Capell A. Multhaup G. Condron M.M. Teplow D.B. Haass C. EMBO Rep. 2001; 2: 835-841Crossref PubMed Scopus (424) Google Scholar, 12Weidemann A. Eggert S. Reinhard F.B. Vogel M. Paliga K. Baier G. Masters C.L. Beyreuther K. Evin G. Biochemistry. 2002; 41: 2825-2835Crossref PubMed Scopus (315) Google Scholar). ϵ-Cleavage produces two APP intracellular domains (AICD), AICD49–99 and AICD50–99. Familial AD mutations in presenilin 1/2 and APP invariably increase the level of AICD49–99, a potential counterpart of Aβ48, as well as the ratio of Aβ42/Aβ40 (13Sato T. Dohmae N. Qi Y. Kakuda N. Misonou H. Mitsumori R. Maruyama H. Koo E.H. Haass C. Takio K. Morishima-Kawashima M. Ishiura S. Ihara Y. J. Biol. Chem. 2003; 278: 24294-24301Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). In addition, the expression of Aβ48 in cultured cells was shown to lead to an increase in the Aβ42/Aβ40 ratio (14Funamoto S. Morishima-Kawashima M. Tanimura Y. Hirotani N. Saido T.C. Ihara Y. Biochemistry. 2004; 43: 13532-13540Crossref PubMed Scopus (115) Google Scholar). Thus ϵ-cleavage may determine the site preference of the γ-cleavage and the final Aβ species.Recently, we established a CHAPSO-solubilized γ-secretase assay system that exhibits high specific activity (15Kakuda N. Funamoto S. Yagishita S. Takami M. Osawa S. Dohmae N. Ihara Y. J. Biol. Chem. 2006; 281: 14776-14786Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). We confirmed that equimolar amounts of Aβ and AICD were produced from C99 by γ-secretase. In a series of experiments, we noted that the concentrations of detergent and phosphatidylcholine significantly affected γ-secretase activity (15Kakuda N. Funamoto S. Yagishita S. Takami M. Osawa S. Dohmae N. Ihara Y. J. Biol. Chem. 2006; 281: 14776-14786Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 16Li 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 (496) Google Scholar, 17Fraering 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 (201) Google Scholar, 18Fraering 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 (118) Google Scholar). This observation points to the possibility that a change in the composition of the lipid bilayer could have a significant impact on the enzymatic activity of γ-secretase. The level of secreted Aβ from cultured cells was reported to be attenuated by increasing levels of phosphatidylinositol 4,5-diphosphate [PI(4,5)P2], one of the phosphoinositides (19Landman N. Jeong S.Y. Shin S.Y. Voronov S.V. Serban G. Kang M.S. Park M.K. Di Paolo G. Chung S. Kim T.W. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 19524-19529Crossref PubMed Scopus (108) Google Scholar). The phosphoinositides play pivotal roles in numerous biological processes, such as ion channel regulation, membrane trafficking, cell polarity, and actin rearrangement (20Brown F.D. Rozelle A.L. Yin H.L. Balla T. Donaldson J.G. J. Cell Biol. 2001; 154: 1007-1017Crossref PubMed Scopus (359) Google Scholar, 21Funamoto S. Meili R. Lee S. Parry L. Firtel R.A. Cell. 2002; 109: 611-623Abstract Full Text Full Text PDF PubMed Scopus (621) Google Scholar, 22Funamoto S. Milan K. Meili R. Firtel R.A. J. Cell Biol. 2001; 153: 795-810Crossref PubMed Scopus (197) Google Scholar, 23Kanzaki M. Furukawa M. Raab W. Pessin J.E. J. Biol. Chem. 2004; 279: 30622-30633Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 24Oliver D. Lien C.C. Soom M. Baukrowitz T. Jonas P. Fakler B. Science. 2004; 304: 265-270Crossref PubMed Scopus (278) Google Scholar, 25Michailidis I.E. Helton T.D. Petrou V.I. Mirshahi T. Ehlers M.D. Logothetis D.E. J. Neurosci. 2007; 27: 5523-5532Crossref PubMed Scopus (44) Google Scholar).PI(4,5)P2 is estimated as 0.3–2.0% of the total cellular lipid. McLaughlin et al. (26McLaughlin S. Wang J. Gambhir A. Murray D. Annu. Rev. Biophys. Biomol. Struct. 2002; 31: 151-175Crossref PubMed Scopus (692) Google Scholar) estimated that the intracellular concentration of PI(4,5)P2, if uniformly distributed inside the cell, was in the range of 2–30 μm, based on the dissociation constants between PH domains and PI(4,5)P2. Bunce et al. (27Bunce C.M. French P.J. Allen P. Mountford J.C. Moor B. Greaves M.F. Michell R.H. Brown G. Biochem. J. 1993; 289: 667-673Crossref PubMed Scopus (79) Google Scholar) reported that the concentration of PI(4,5)P2 was in the range of 32–159 μm in several cell species. Even though the phosphoinositides are a minor component of cellular lipids, one can assume that their concentrations in the two-dimensional intact membrane would be higher than that reported for the total three-dimensional cell volume. If the volume of the membrane is estimated as ∼10–20% of the cell volume, the concentrations of PI(4,5)P2 in the membrane are at least five times higher than those in the total cell volume. The local concentration of PI(4,5)P2 at the inner leaflet of neutrophil membrane was reported to be ∼5 mm in the steady state (28Lemmon M.A. Ferguson K.M. Biochem. J. 2000; 350: 1-18Crossref PubMed Scopus (613) Google Scholar). Further, Sheetz et al. (29Sheetz M.P. Febbroriello P. Koppel D.E. Nature. 1982; 296: 91-93Crossref PubMed Scopus (38) Google Scholar, 30Sheetz M.P. Nat. Rev. Mol. Cell Biol. 2001; 2: 392-396Crossref PubMed Scopus (358) Google Scholar) showed that the concentration of PI(4,5)P2 was 4–8 mm in a 50-Å area of the inner leaflet of erythrocyte membrane. In addition, a number of reports showed that PI(4,5)P2 localizes in the detergent-insoluble microdomains (rafts) of the membrane (31Laux T. Fukami K. Thelen M. Golub T. Frey D. Caroni P. J. Cell Biol. 2000; 149: 1455-1472Crossref PubMed Scopus (513) Google Scholar, 32Rozelle A.L. Machesky L.M. Yamamoto M. Driessens M.H. Insall R.H. Roth M.G. Luby-Phelps K. Marriott G. Hall A. Yin H.L. Curr. Biol. 2000; 10: 311-320Abstract Full Text Full Text PDF PubMed Scopus (429) Google Scholar, 33Golub T. Caroni P. J. Cell Biol. 2005; 169: 151-165Crossref PubMed Scopus (131) Google Scholar). It has been proposed that there is a spatially confined pool of PI(4,5)P2 in the membrane (34Simonsen A. Wurmser A.E. Emr S.D. Stenmark H. Curr. Opin. Cell Biol. 2001; 13: 485-492Crossref PubMed Scopus (405) Google Scholar, 35Martin T.F. Curr. Opin. Cell Biol. 2001; 13: 493-499Crossref PubMed Scopus (327) Google Scholar, 36Hinchliffe K.A. Ciruela A. Irvine R.F. Biochim. Biophys. Acta. 1998; 1436: 87-104Crossref PubMed Scopus (102) Google Scholar, 37Janmey P.A. Lindberg U. Nat. Rev. Mol. Cell Biol. 2004; 5: 658-666Crossref PubMed Scopus (184) Google Scholar). Thus it is reasonable to consider that the concentration of PI(4,5)P2 in the microdomains of the membrane is much higher than previously thought. γ-Secretase is also enriched in lipid raft microdomains (38Vetrivel K.S. Cheng H. Kim S.H. Chen Y. Barnes N.Y. Parent A.T. Sisodia S.S. Thinakaran G. J. Biol. Chem. 2005; 280: 25892-25900Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar, 39Vetrivel K.S. Cheng H. Lin W. Sakurai T. Li T. Nukina N. Wong P.C. Xu H. Thinakaran G. J. Biol. Chem. 2004; 279: 44945-44954Abstract Full Text Full Text PDF PubMed Scopus (360) Google Scholar, 40Wada S. Morishima-Kawashima M. Qi Y. Misono H. Shimada Y. Ohno-Iwashita Y. Ihara Y. Biochemistry. 2003; 42: 13977-13986Crossref PubMed Scopus (71) Google Scholar). It is likely that the phosphoinositides and γ-secretase localize in the same membrane microdomains.Furthermore, phosphoinositides were widely known to be modulated by their concentrations in membrane by extracellular stimulus in physiological condition (41Stephens L.R. Hughes K.T. Irvine R.F. Nature. 1991; 351: 33-39Crossref PubMed Scopus (386) Google Scholar). Studies of several PH domains fused with green fluorescent protein revealed that concentrations of PI(3,4)P2 and PI(3,4,5)P3 were elevated after stimulus in Dictyostelium cells and neutrophils in vivo (21Funamoto S. Meili R. Lee S. Parry L. Firtel R.A. Cell. 2002; 109: 611-623Abstract Full Text Full Text PDF PubMed Scopus (621) Google Scholar, 22Funamoto S. Milan K. Meili R. Firtel R.A. J. Cell Biol. 2001; 153: 795-810Crossref PubMed Scopus (197) Google Scholar, 42Wang F. Herzmark P. Weiner O.D. Srinivasan S. Servant G. Bourne H.R. Nat. Cell Biol. 2002; 4: 513-518Crossref PubMed Scopus (395) Google Scholar, 43Servant G. Weiner O.D. Herzmark P. Balla T. Sedat J.W. Bourne H.R. Science. 2000; 287: 1037-1040Crossref PubMed Scopus (728) Google Scholar, 44Rickert P. Weiner O.D. Wang F. Bourne H.R. Servant G. Trends Cell Biol. 2000; 10: 466-473Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar). In neutrophils, it has been reported that the local concentration of PI(3,4,5)P3 at the inner leaflet of the plasma membrane is 5 μm and that after extracellular stimulation it increases to 200 μm (28Lemmon M.A. Ferguson K.M. Biochem. J. 2000; 350: 1-18Crossref PubMed Scopus (613) Google Scholar). The concentration of PI(3,4)P2 is estimated to increase from 10–20 to 100–200 μm upon stimulation (28Lemmon M.A. Ferguson K.M. Biochem. J. 2000; 350: 1-18Crossref PubMed Scopus (613) Google Scholar). Winks et al. (45Winks J.S. Hughes S. Filippov A.K. Tatulian L. Abogadie F.C. Brown D.A. Marsh S.J. J. Neurosci. 2005; 25: 3400-3413Crossref PubMed Scopus (143) Google Scholar) reported that concentration of PI(4,5)P2 increased from 192–381 to 417–1153 μm after expression of PI5K in superior cervical ganglia. Those observations suggest that concentration of phosphoinositides can be modulated in physiological conditions. Recently, increasing phosphoinositide (PI(4,5)P2) levels alter Aβ production by cultured cells (19Landman N. Jeong S.Y. Shin S.Y. Voronov S.V. Serban G. Kang M.S. Park M.K. Di Paolo G. Chung S. Kim T.W. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 19524-19529Crossref PubMed Scopus (108) Google Scholar), implying a cross-talk between phosphoinositides and γ-secretase. Here we examined whether there are direct effects of phosphoinositides on γ-secretase in both CHAPSO-soluble and -insoluble states.EXPERIMENTAL PROCEDURESCell Culture—Chinese hamster ovary (CHO) cells were cultured in Dulbecco's modified Eagle's medium (Sigma) containing 10% fetal bovine serum (Invitrogen) and penicillin/streptomycin (Invitrogen). Stable T-Rex-CHO cells (Invitrogen) inducibly expressing C99 were grown in F-12 nutrient mixture (Invitrogen) containing 10% fetal bovine serum (Invitrogen), penicillin/streptomycin, 250 μg/ml Zeocin (Invitrogen), and 10 μg/ml Blasticidine S (Invitrogen) (46Qi-Takahara Y. Morishima-Kawashima M. Tanimura Y. Dolios G. Hirotani N. Horikoshi Y. Kametani F. Maeda M. Saido T.C. Wang R. Ihara Y. J. Neurosci. 2005; 25: 436-445Crossref PubMed Scopus (307) Google Scholar).γ-Secretase Assay and Aβ Quantification—Microsomal fractions of CHO cells were obtained as previously described (15Kakuda N. Funamoto S. Yagishita S. Takami M. Osawa S. Dohmae N. Ihara Y. J. Biol. Chem. 2006; 281: 14776-14786Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar) and solubilized on ice by the addition of equal volumes of 2× NK buffer (50 mm PIPES, pH 7.2, 250 mm sucrose, 1 mm EGTA, 2% CHAPSO, 1 mm diisopropyl phosphorofluoridate, 20 μg/ml antipain, 20 μg/ml leupeptin, 10 μg/ml TLCK, 10 mm phenanthroline, and 2 mm thiorphan). The supernatant obtained after 100,000 × g centrifugation for 1 h was diluted with three volumes of the dilution buffer (50 mm PIPES, pH 7.2, 0.166% CHAPSO, 250 mm sucrose, 1 mm EGTA, 1 mm diisopropyl phosphorofluoridate, 10 μg/ml antipain, 10 μg/ml leupeptin, 10 μg/ml TLCK, 5 mm phenanthroline, 1 mm thiorphan, 1.33 μm pepstatin A, and 0.133% phosphatidylcholine). The diluted supernatants contained a final concentration of 0.1% (equivalent to 1.3 mm) phosphatidylcholine and 0.375% CHAPSO unless otherwise indicated. Defined amounts of C99-FLAG substrate were incubated with the CHAPSO lysate at 37 °C for 4 h (15Kakuda N. Funamoto S. Yagishita S. Takami M. Osawa S. Dohmae N. Ihara Y. J. Biol. Chem. 2006; 281: 14776-14786Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). We observed that the addition of 1 μm pepstatin A prevented γ-secretase-independent C99-FLAG cleavage. Incubated reaction mixtures were subjected to Western blotting for Aβ quantification, as described (14Funamoto S. Morishima-Kawashima M. Tanimura Y. Hirotani N. Saido T.C. Ihara Y. Biochemistry. 2004; 43: 13532-13540Crossref PubMed Scopus (115) Google Scholar). After Aβ transfer, the nitrocellulose membrane was boiled for 5 min in the aluminum boiling apparatus (Can Do, Tokyo, Japan) for enhanced detection. Aβ on the membrane were visualized by an ECL system (GE Healthcare) using the well characterized monoclonal antibodies 82E1, BA27 (highly specific for the Aβ40 carboxyl terminus), and BC05 (raised against Aβ35–43, specific for the Aβ42 carboxyl terminus, but cross-reactive with CTFs and full-length APP), for assessing the total Aβ, Aβ40, and Aβ42, respectively (47Suzuki N. Cheung T.T. Cai X.D. Odaka A. Otvos Jr., L. Eckman C. Golde T.E. Younkin S.G. Science. 1994; 264: 1336-1340Crossref PubMed Scopus (1338) Google Scholar).Preparation of Notch Substrate—For assessment of γ-secretase-dependent Notch S3 cleavage in the presence of phosphoinositides, we expressed an artificial Notch substrate with shortened intracellular domain (designated as ΔE Notch-FLAG) in Sf9 cells (48Saxena M.T. Schroeter E.H. Mumm J.S. Kopan R. J. Biol. Chem. 2001; 276: 40268-40273Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). Isolated Notch substrate was incubated with the CHAPSO lysate at 37 °C for 4 h (15Kakuda N. Funamoto S. Yagishita S. Takami M. Osawa S. Dohmae N. Ihara Y. J. Biol. Chem. 2006; 281: 14776-14786Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar) (supplemental Fig. S1A). γ-Secretase-dependent S3 cleavage was visualized by detecting shortened Notch intracellular domain fused with FLAG tag (sNICD-FLAG) with ANTI-FLAG® M2 monoclonal antibody (Sigma) (supplemental Fig. S1B). We confirmed that ΔE Notch-FLAG was cleaved at the bona fide S3 cleavage site by γ-secretase (see supplemental Fig. S1, C and D).Isolation of CHAPSO-insoluble Rafts—The CHAPSO-insoluble fraction was obtained as described previously (40Wada S. Morishima-Kawashima M. Qi Y. Misono H. Shimada Y. Ohno-Iwashita Y. Ihara Y. Biochemistry. 2003; 42: 13977-13986Crossref PubMed Scopus (71) Google Scholar). The T-Rex-CHO stable cell line was cultured in the presence of tetracycline to induce expression of C99 (46Qi-Takahara Y. Morishima-Kawashima M. Tanimura Y. Dolios G. Hirotani N. Horikoshi Y. Kametani F. Maeda M. Saido T.C. Wang R. Ihara Y. J. Neurosci. 2005; 25: 436-445Crossref PubMed Scopus (307) Google Scholar). Microsomal fractions of the cells were homogenized in five volumes of 10% sucrose in MES-buffered saline (25 mm MES, pH 6.5, and 150 mm NaCl) containing 1% CHAPSO. After adjusting the sucrose concentration to 40%, the homogenate was centrifuged on a discontinuous sucrose gradient (5, 35, and 40%) at 39,000 rpm for 20 h at 4 °C on an SW 41 Ti rotor (Beckman). The interface between 5 and 35% sucrose was collected and designated as CHAPSO-insoluble rafts. The CHAPSO-insoluble rafts were diluted with three volumes of dilution buffer (20 mm PIPES, pH 7.2, 140 mm KCl, 250 mm sucrose, 5 mm EGTA) and incubated for 45 min at 37 °C in the presence or absence of PI(4,5)P2. For assessing Aβ production from exogenously added C99-FLAG, the CHAPSO insoluble rafts that were obtained from cells grown in the absence of tetracycline were incubated for 60 min with 100 nm C99-FLAG in the presence or absence of PI(4,5)P2.Treatment of PLC Inhibitor—7WD10, CHO cells expressing APP751 were cultured in Dulbecco's modified Eagle's medium (Sigma) containing 10% fetal bovine serum (Invitrogen) and 200 μg/ml G418 (49Koo E.H. Squazzo S.L. J. Biol. Chem. 1994; 269: 17386-17389Abstract Full Text PDF PubMed Google Scholar). The cells were treated with phosphatidylinositol specific PLC inhibitor, edelfosin (Calbiochem) at a concentration of 15 μm in the absence of G418 for 6 h. Microsomal fraction of the cells were prepared as described previously (15Kakuda N. Funamoto S. Yagishita S. Takami M. Osawa S. Dohmae N. Ihara Y. J. Biol. Chem. 2006; 281: 14776-14786Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). 500 μl of the microsomal fraction (2.5 mg/ml protein concentration) was mixed with 1 ml of MeOH:CHCl3 (2:1) and centrifuged at 15,000 rpm for 5 min at 4 °C. Resultant pellet was mixed with the same solvent to complete neutral lipids extraction. 750 μl of MeOH: CHCl3:12 N HCl (40:80:1) was added to the pellet and mixed for extraction of acidic lipids. Supernatant was transferred to a new 1.5-ml tube and mixed with 250 μl of CHCl3 and 450 μl of 0.1 n HCl. After centrifugation, the organic phase was transferred to a new tube and dried up. The dried lipid sample was reconstituted with 80 μl of CHCl3: MeOH:H2O (1:2:0.8) and spotted onto PI(4,5)P2 Mass Strip (Echelon). PI(4,5)P2 in extracted lipid sample was detected with PLC-δ1 PH domain glutathione S-transferase-tagged protein (Echelon). Miocrosomal fraction prepared from cells treated with edelfosin was incubated at 37 °C for 0.5 h in the presence of 15 μm edelfosin and subjected to Western blot to assess effect of edelfosin on Aβ production from isolated membrane (10Gu Y. Misonou H. Sato T. Dohmae N. Takio K. Ihara Y. J. Biol. Chem. 2001; 276: 35235-35238Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar, 13Sato T. Dohmae N. Qi Y. Kakuda N. Misonou H. Mitsumori R. Maruyama H. Koo E.H. Haass C. Takio K. Morishima-Kawashima M. Ishiura S. Ihara Y. J. Biol. Chem. 2003; 278: 24294-24301Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar).Phospholipids and Derivatives— l-α-Phosphatidylcholine (PC) was purchased from Sigma, dissolved in 1% CHAPSO solution, and stored as such. d-myo-inositol 1,4,5-triphosphate from Sigma was dissolved in water to 19.6 mm stock solution. PI C-16, phosphatidylinositol 3-phosphate C-16, phosphatidylinositol 4-phosphate C-16, phosphatidylinositol 5-phosphate C-16 (PI(5)P), phosphatidylinositol 3,4-diphosphate C-16 (PI(3,4)P2), phosphatidylinositol 4,5-diphosphate C-16 (PI(4,5)P2), and phosphatidylinositol 3,4,5-triphosphate C-16 (PI(3,4,5)P3) from Cayman Chemical were dissolved in 0.25% CHAPSO and stored as 8.45 mm solutions. It is essential to avoid multiple freezing and thawing of these stock solutions. Stock solutions were repackaged into smaller vials and stored at –20 °C. Defined amounts of phospholipids and derivatives were mixed with γ-secretase reaction mixtures for incubation as above.Immunoprecipitation—γ-Secretase complex was immunoprecipitated with anti-nicastrin polyclonal antibody (Sigma), as previously described (15Kakuda N. Funamoto S. Yagishita S. Takami M. Osawa S. Dohmae N. Ihara Y. J. Biol. Chem. 2006; 281: 14776-14786Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). After thorough washing, the γ-secretase complex bound to protein A-Sepharose beads was incubated in 0.25% CHAPSO buffer (50 mm PIPES, pH 7.2, 250 mm sucrose, 1 mm EGTA, 0.25% CHAPSO, 2 mm diisopropyl phosphorofluoridate, 20 μg/ml antipain, 20 μg/ml leupeptin, 20 μg/ml TLCK, 10 mm phenanthroline, 2 mm thiorphan, and 0.1% phosphatidylcholine) with C99-FLAG substrate at 37 °C for 4 h together with defined concentrations of PI(4,5)P2. To evaluate inhibitory effects of PI(4,5)P2 on the interaction between γ-secretase and C99-FLAG substrate, C99-FLAG prebound anti-FLAG M2 agarose beads (Sigma) were mixed with the CHAPSO-solubilized microsomal fraction of CHO cells and incubated at 4 °C overnight in the presence or absence of 0.845 mm PI(4,5)P2. The agarose beads were washed three times and subjected to Western blotting to visualize γ-secretase components, including nicastrin, carboxyl-terminal fragment (CTF) of presenilin 1, and Aph-1. Presenilin 1 CTF, Aph-1, and Pen-2 were detected with anti-presenilin 1 CTF antiserum (a gift from Dr. Iwatsubo, University of Tokyo), anti-Aph1 polyclonal antibody (Covance), and anti-Pen-2 polyclonal antibody (50Shimojo M. Sahara N. Murayama M. Ichinose H. Takashima A. Neurosci. Res. 2007; 57: 446-453Crossref PubMed Scopus (22) Google Scholar), respectively.RESULTSEffects of PI(4,5)P2 on γ-Secretase—To demonstrate the effects of phosphoinositides on γ-secretase activity, a CHAPSO-solubilized γ-secretase assay was performed in the presence of various concentrations of PI(4,5)P2 (15Kakuda N. Funamoto S. Yagishita S. Takami M. Osawa S. Dohmae N. Ihara Y. J. Biol. Chem. 2006; 281: 14776-14786Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). The addition of PC to the CHAPSO-solubilized γ-secretase reaction mixture at increasing concentrations up to 1.3 mm (equivalent to 0.1%) enhanced the production of Aβ as described previously (15Kakuda N. Funamoto S. Yagishita S. Takami M. Osawa S. Dohmae N. Ihara Y. J. Biol. Chem. 2006; 281: 14776-14786Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 17Fraering 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 (201) Google Scholar, 18Fraering 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 (118) Google Scholar) (Fig. 1A), whereas increasing concentrations of PI(4,5)P2 dramatically reduced Aβ production with an IC50 of 141 μm (equivalent to 0.016%) (Fig. 1B). The inhibitory effect of PI(4,5)P2 was observed even in the presence of 0.1% (1.3 mm) PC, with the IC50 being ∼551 μm (equivalent to 0.06%) (Fig. 1C). We observed that PI(4,5)P2 suppressed AICD and Aβ production in parallel (data not shown). To further confirm a direct effect of PI(4,5)P2 on γ-secretase activity, t" @default.
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• W2040137618 title "Phosphoinositides Suppress γ-Secretase in Both the Detergent-soluble and -insoluble States" @default.
• W2040137618 cites W1502820594 @default.
• W2040137618 cites W1560202903 @default.
• W2040137618 cites W1856515523 @default.
• W2040137618 cites W1963904512 @default.
• W2040137618 cites W1977412572 @default.
• W2040137618 cites W1978285772 @default.
• W2040137618 cites W1980906872 @default.
• W2040137618 cites W1998931340 @default.
• W2040137618 cites W2003890494 @default.
• W2040137618 cites W2004465970 @default.
• W2040137618 cites W2005873882 @default.
• W2040137618 cites W2009756576 @default.
• W2040137618 cites W2013163003 @default.
• W2040137618 cites W2016441216 @default.
• W2040137618 cites W2021989107 @default.
• W2040137618 cites W2022114463 @default.
• W2040137618 cites W2023628337 @default.
• W2040137618 cites W2023712216 @default.
• W2040137618 cites W2027044703 @default.
• W2040137618 cites W2028673763 @default.
• W2040137618 cites W2033796514 @default.
• W2040137618 cites W2034146282 @default.
• W2040137618 cites W2036887265 @default.
• W2040137618 cites W2039999119 @default.
• W2040137618 cites W2043420241 @default.
• W2040137618 cites W2046414016 @default.
• W2040137618 cites W2047442659 @default.
• W2040137618 cites W2049451196 @default.
• W2040137618 cites W2055717995 @default.
• W2040137618 cites W2056790763 @default.
• W2040137618 cites W2057709960 @default.
• W2040137618 cites W2067216245 @default.
• W2040137618 cites W2067772115 @default.
• W2040137618 cites W2070189344 @default.
• W2040137618 cites W2075829521 @default.
• W2040137618 cites W2088532612 @default.
• W2040137618 cites W2088881960 @default.
• W2040137618 cites W2092249847 @default.
• W2040137618 cites W2097750046 @default.
• W2040137618 cites W2100784365 @default.
• W2040137618 cites W2101351668 @default.
• W2040137618 cites W2107551117 @default.
• W2040137618 cites W2109040420 @default.
• W2040137618 cites W2118330889 @default.
• W2040137618 cites W2119806797 @default.
• W2040137618 cites W2123332655 @default.
• W2040137618 cites W2124097986 @default.
• W2040137618 cites W2128084592 @default.
• W2040137618 cites W2133019124 @default.
• W2040137618 cites W2134283668 @default.
• W2040137618 cites W2137072938 @default.
• W2040137618 cites W2151462825 @default.
• W2040137618 cites W2151519283 @default.
• W2040137618 cites W2169152986 @default.
• W2040137618 cites W2171857030 @default.
• W2040137618 cites W4238658571 @default.
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