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• W1978281908 abstract "γ-Secretase cleaves the transmembrane domain of β-amyloid precursor protein at multiple sites referred to as γ-, ϵ-, and ζ-cleavage sites. We previously showed that N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT), a potent dipeptide γ-secretase inhibitor, causes differential accumulation of longer amyloid β-proteins (Aβs) within Chinese hamster ovary cells co-expressing β C-terminal fragment and wild-type presenilin 1 (C99/wtPS1 cells). In this study, we used sucrose density gradient centrifugation to fractionate the membranes from C99/wtPS1 cells that had been pretreated with DAPT. We found that accumulating Aβ46 localized exclusively to low density membrane (LDM) domains. Incubating the Aβ46-accumulating LDM domains at 37 °C produced Aβ40, Aβ42, Aβ43, and β-amyloid precursor protein intracellular domain. The addition of L685,458 completely prevented β-amyloid precursor protein intracellular domain generation and resulted in a large decrease in the level of Aβ46 and the concomitant appearance of Aβ40 and Aβ43 but not Aβ42. Further addition of DAPT suppressed the production of Aβ40/43 and abolished the decrease in the amount of Aβ46. These data indicate that preaccumulated Aβ46 is processed by γ-secretase to Aβ40/43 but not to Aβ42 in the LDM domains. The amount of newly produced Aβ40 and Aβ43 was roughly equivalent to the decrease in the amount of Aβ46. Temporal profiles did not show a maximal concentration for Aβ43, suggesting that Aβ46 is processed to Aβ40 and Aβ43 through a nonsuccessive process. γ-Secretase cleaves the transmembrane domain of β-amyloid precursor protein at multiple sites referred to as γ-, ϵ-, and ζ-cleavage sites. We previously showed that N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT), a potent dipeptide γ-secretase inhibitor, causes differential accumulation of longer amyloid β-proteins (Aβs) within Chinese hamster ovary cells co-expressing β C-terminal fragment and wild-type presenilin 1 (C99/wtPS1 cells). In this study, we used sucrose density gradient centrifugation to fractionate the membranes from C99/wtPS1 cells that had been pretreated with DAPT. We found that accumulating Aβ46 localized exclusively to low density membrane (LDM) domains. Incubating the Aβ46-accumulating LDM domains at 37 °C produced Aβ40, Aβ42, Aβ43, and β-amyloid precursor protein intracellular domain. The addition of L685,458 completely prevented β-amyloid precursor protein intracellular domain generation and resulted in a large decrease in the level of Aβ46 and the concomitant appearance of Aβ40 and Aβ43 but not Aβ42. Further addition of DAPT suppressed the production of Aβ40/43 and abolished the decrease in the amount of Aβ46. These data indicate that preaccumulated Aβ46 is processed by γ-secretase to Aβ40/43 but not to Aβ42 in the LDM domains. The amount of newly produced Aβ40 and Aβ43 was roughly equivalent to the decrease in the amount of Aβ46. Temporal profiles did not show a maximal concentration for Aβ43, suggesting that Aβ46 is processed to Aβ40 and Aβ43 through a nonsuccessive process. Amyloid β-protein (Aβ) 2The abbreviations used are:Aβamyloid β-proteinADAlzheimer diseaseAPPβ-amyloid precursor proteinAICDAPP intracellular domainCHOChinese hamster ovaryCTFC-terminal fragmentDAPTN-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl esterFADfamilial Alzheimer diseaseLDMlow density membraneMES4-morpholineethanesulfonic acidMBSMES-buffered salinemtmutantPSpresenilinwtwild typeCHAPSO3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonic acidPIPES1,4-piperazinediethanesulfonic acidTricineN-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. 2The abbreviations used are:Aβamyloid β-proteinADAlzheimer diseaseAPPβ-amyloid precursor proteinAICDAPP intracellular domainCHOChinese hamster ovaryCTFC-terminal fragmentDAPTN-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl esterFADfamilial Alzheimer diseaseLDMlow density membraneMES4-morpholineethanesulfonic acidMBSMES-buffered salinemtmutantPSpresenilinwtwild typeCHAPSO3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonic acidPIPES1,4-piperazinediethanesulfonic acidTricineN-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. is the major component of the senile plaque, a neuropathological hallmark of Alzheimer disease (AD). Aβ is produced from β-amyloid precursor protein (APP), through sequential cleavages by two membrane proteases referred to as β- and γ-secretases (1Selkoe D.J. Physiol. Rev. 2001; 81: 741-766Crossref PubMed Scopus (5092) Google Scholar). β-Secretase, or β-site APP-cleaving enzyme 1 (2Vassar 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 (3235) Google Scholar), is a membrane-bound aspartyl protease that cleaves APP in its luminal region, generating a 99-residue fragment (C99) called β C-terminal fragment (βCTF). βCTF in turn is cleaved in the middle of its transmembrane domain by γ-secretase, releasing Aβ and APP intracellular domain (AICD). Accumulating evidence strongly suggests that γ-secretase is also an aspartyl protease of high molecular weight protein complex (3Wolfe M.S. Haass C. J. Biol. Chem. 2001; 276: 5413-5416Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar), which includes at least four distinct membrane proteins, presenilin (PS) 1 or 2, nicastrin, Aph-1, and Pen-2. In particular, PS1 or PS2 is believed to compose the catalytic site(s) of γ-secretase (4Kimberly 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 (673) 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 (772) Google Scholar, 6Edbauer D. Winkler E. Regula J.T. Pesold B. Steiner H. Haass C. Nat. Cell Biol. 2003; 5: 486-488Crossref PubMed Scopus (767) Google Scholar). amyloid β-protein Alzheimer disease β-amyloid precursor protein APP intracellular domain Chinese hamster ovary C-terminal fragment N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester familial Alzheimer disease low density membrane 4-morpholineethanesulfonic acid MES-buffered saline mutant presenilin wild type 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonic acid 1,4-piperazinediethanesulfonic acid N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. amyloid β-protein Alzheimer disease β-amyloid precursor protein APP intracellular domain Chinese hamster ovary C-terminal fragment N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester familial Alzheimer disease low density membrane 4-morpholineethanesulfonic acid MES-buffered saline mutant presenilin wild type 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonic acid 1,4-piperazinediethanesulfonic acid N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. Whereas the most abundantly secreted Aβ species is Aβ40, the minor, two-residue longer one, Aβ42, is by far the predominant species deposited in senile plaques (7Iwatsubo T. Odaka A. Suzuki N. Mizusawa H. Nukina N. Ihara Y. Neuron. 1994; 13: 45-53Abstract Full Text PDF PubMed Scopus (1523) Google Scholar). Thus far, three causative genes for familial AD (FAD), APP, PS1, and PS2, have been identified, and the FAD mutations on those genes increase the proportion of Aβ42 relative to the total Aβ produced (1Selkoe D.J. Physiol. Rev. 2001; 81: 741-766Crossref PubMed Scopus (5092) Google Scholar). Because Aβ42 production or deposition is the key factor in the development of AD, the regulatory mechanisms of intramembrane cleavage at Aβ40 and Aβ42 sites (γ-cleavage sites) are a pivotal issue for understanding γ-secretase. Besides the γ-cleavage sites, cleavage sites near the membrane-cytoplasm boundary (ϵ-cleavage sites) have been identified by sequencing AICD produced by the cell-free Aβ and AICD generation system (8Gu 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 (266) Google Scholar). Most unexpectedly, the two AICD species generated in vitro do not start from position 41 or 43; the major species is AICD-(50–99), and the minor species is AICD-(49–99). FAD mutations on APP and PS1/2 increase the proportion of AICD-(49–99) and Aβ42 (9Sato 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). This suggests some relationship between Aβ42 and AICD-(49–99) and between Aβ40 and AICD-(50–99). Failure to detect any longer AICD and the presence of a variety of longer Aβs suggest that ϵ-cleavage precedes γ-cleavage (8Gu 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 (266) Google Scholar, 10Qi-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 (302) Google Scholar), and this involves a stepwise processing of longer Aβs (10Qi-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 (302) Google Scholar). Within cells, the expression of Aβ49 and Aβ48, the postulated counterparts generated by ϵ-cleavage, results in preferentially Aβ40 and Aβ42 production, respectively (11Funamoto S. Morishima-Kawashima M. Tanimura Y. Hirotani N. Saido T.C. Ihara Y. Biochemistry. 2004; 43: 13532-13540Crossref PubMed Scopus (113) Google Scholar). Moreover, the potent dipeptide γ-secretase inhibitor N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT) causes intracellular accumulation of longer Aβ species, Aβ43 and Aβ46, which differentially accumulate in the Chinese hamster ovary (CHO) cells expressing wild-type (wt) PS1. In contrast, Aβ45 and Aβ48 accumulate in the CHO cells expressing mutant (mt) PS2 (N141I) and mtPS1 (M233T), respectively (10Qi-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 (302) Google Scholar, 12Yagishita S. Morishima-Kawashima M. Tanimura Y. Ishiura S. Ihara Y. Biochemistry. 2006; 45: 3952-3960Crossref PubMed Scopus (54) Google Scholar). These observations led us to speculate that this process occurs through a stepwise (successive) processing mediated by γ-secretase of βCTF at every third residue. According to this model, Aβ40 should be generated through Aβ43/46 from Aβ49 generated by ϵ-cleavage. To test this model, we examined whether Aβ46 is processed to shorter Aβs in the low density membrane (LDM) domains. Cell Culture—CHO cells co-expressing wtPS1 and βCTF (C99/wtPS1 cells) (12Yagishita S. Morishima-Kawashima M. Tanimura Y. Ishiura S. Ihara Y. Biochemistry. 2006; 45: 3952-3960Crossref PubMed Scopus (54) Google Scholar) were maintained in F-12 nutrient mixture (Invitrogen) containing 10% fetal calf serum (Invitrogen), penicillin/streptomycin, 250 μg/ml Zeocin (Invitrogen), 10 μg/ml blasticidin S (Invitrogen), and 200 μg/ml G418 (Calbiochem). In these cells, βCTF was inducibly expressed in the presence of 1 μg/ml tetracycline (Invitrogen). Antibodies—Monoclonal antibodies to Aβ used in this study were 82E1 (highly specific for Asp-1 of human Aβ) (IBL, Fujioka, Japan), BA27 (raised against Aβ-(1–40), highly specific for Aβ40), and BC05 (raised against Aβ-(35–43), specific for Aβ42). A polyclonal antibody UT421 (raised against the 20-residue cytoplasmic domain of APP; a gift of Dr. T. Suzuki, Hokkaido University) was used to detect βCTF and AICD. Monoclonal antibodies to caveolin, flotillin, and calnexin were purchased from BD Biosciences. Treatment with γ-Secretase Inhibitors—The γ-secretase inhibitors were {1S-benzyl-4R-[1S-carbamoyl-2-phenylethylcarbamoyl-1S-3-methylbutylcarbamoyl]-2R-hydroxy-5-phenylpentyl} carbamic acid tert-butyl ester (L685,458) (13Shearman 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 (364) Google Scholar) and N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT) (14Dovey H.F. John V. Anderson J.P. 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 (787) Google Scholar). Both were purchased from Calbiochem. C99/wtPS1 cells were cultured for 2 h in the presence of 0.25 μm DAPT, and 1 μg/ml tetracycline was added. The cells were harvested with phosphate-buffered saline washes 6 h later. Isolation of CHAPSO-insoluble LDM Domains—The total membrane fraction was obtained as described previously (9Sato 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). Briefly, harvested cells were homogenized in buffer A (20 mm PIPES, pH 7.0, 140 mm KCl, 250 mm sucrose, 5 mm EGTA), and the homogenate was centrifuged at 800 × g for 10 min to remove nuclei and cell debris. The resulting supernatant was centrifuged at 100,000 × g for 1 h. The pellet was suspended in buffer A, and the suspension was centrifuged again. The resulting pellet (the total membrane fraction) was homogenized in 10% sucrose in MES-buffered saline (MBS: 25 mm MES, pH 6.5, 150 mm NaCl) containing 1% CHAPSO and various protease inhibitors. The homogenate was adjusted to 40% sucrose (4 ml) by adding an equal volume of 70% sucrose in MBS, placed at the bottom of an ultracentrifuge tube, and overlaid with 4 ml of 35% sucrose and 4 ml of 5% sucrose in MBS. The discontinuous gradient was centrifuged at 39,000 rpm for 20 h at 4 °C on an SW41 Ti rotor (Beckman, Palo Alto, CA). The interface of the 5/35% sucrose layers (fraction 2), each layer consisting of 5, 35, and 40% sucrose (fractions 1, 3, and 4, respectively), and the pellet (fraction P) were collected carefully. An aliquot from each fraction was subjected to Western blotting. Fraction 2 was diluted with buffer B (50 mm PIPES, pH 7.0, 250 mm sucrose, 1 mm EGTA) and centrifuged. The resulting pellet was washed with buffer B, resuspended in buffer B, and stored at –80 °C. An aliquot from the suspension was subjected to protein determination. Aβ Production by LDM Domains—LDM domains at a protein concentration of 150 μg/ml in buffer B containing various protease inhibitors were incubated at 25 or 37 °C for the times indicated. The reaction was terminated by placing the sample tube on ice and immediately adding chloroform/methanol (2/1). After extraction of lipids with chloroform/methanol, the protein residues were extracted with formic acid, and the extracts were subjected to quantitative Western blotting. Western Blotting—To assess the levels of Aβ or AICD, the samples were run on conventional 16.5% acrylamide Tris/Tricine gels and subjected to Western blotting with 82E1, BA27, BC05, or UT421. To distinguish Aβ species, the samples were separated on SDS/urea gels as described previously, followed by Western blotting with 82E1 (12Yagishita S. Morishima-Kawashima M. Tanimura Y. Ishiura S. Ihara Y. Biochemistry. 2006; 45: 3952-3960Crossref PubMed Scopus (54) Google Scholar). In this study, 11 and 12.5% acrylamide separation gels containing 8 m urea, pH 8.45, were used. The blots were developed by an enhanced chemiluminescence system, and intensities of the signals were quantified using an LAS-1000plus luminescent image analyzer (Fuji Film, Tokyo, Japan). Other Methods—Protein concentrations were determined using the bicinchoninic acid protein assay reagent (Pierce). Predominant Accumulation of Aβ46 in LDM Domains—LDM domains, the cholesterol- and sphingolipid-enriched membrane microdomains, also known as rafts, are assumed to be a site for Aβ production within the cells (15Wada 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). Several proteins involved in Aβ production, such as APP, Aβ (16Morishima-Kawashima M. Ihara Y. Biochemistry. 1998; 37: 15247-15253Crossref PubMed Scopus (150) Google Scholar), β-site APP-cleaving enzyme 1 (17Riddell D.R. Christie G. Hussain I. Dingwall C. Curr. Biol. 2001; 11: 1288-1293Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar), and the four components of γ-secretase (18Vetrivel 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 (359) Google Scholar), 3S. Wada-Kakuda, M. Morishima-Kawashima, and Y. Ihara, unpublished observations. are found in the LDM domains. In particular, active γ-secretase components such as highly glycosylated mature nicastrin and Pen-2 (data not shown) appear to be enriched in the LDM domains (supplemental Fig. S1), and the LDM domains prepared in the presence of CHAPSO exhibit higher γ-secretase activity (15Wada 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, 19Wahrle S. Das P. Nyborg A.C. McLendon C. Shoji M. Kawarabayashi T. Younkin L.H. Younkin S.G. Golde T.E. Neurobiol. Dis. 2002; 9: 11-23Crossref PubMed Scopus (355) Google Scholar). Because our previous study showed that treatment of C99/wtPS1 cells with a higher dose of DAPT causes intracellular accumulation of Aβ46 (12Yagishita S. Morishima-Kawashima M. Tanimura Y. Ishiura S. Ihara Y. Biochemistry. 2006; 45: 3952-3960Crossref PubMed Scopus (54) Google Scholar), we examined where the accumulating Aβ46 localizes in the cell. The total membrane fraction was obtained from DAPT-treated C99/wtPS1 cells and subjected to sucrose density gradient centrifugation in the presence of 0.5% CHAPSO (see “Experimental Procedures”). Fraction 2 from the 5/35% interface represents the LDM domains and is characterized by the presence of caveolin and flotillin, well known markers for LDM domains (Fig. 1A). Aβ46 that accumulated within the cells localized exclusively to LDM domains (fraction 2), whereas βCTF and very small amounts of the remaining Aβ40 (and Aβ42) were partitioned almost equally to the LDM domains (fraction 2) and the cytosolic fraction (fraction 4) (Fig. 1, B and C). Aβ46 Is Processed to Aβ40/43 but Not to Aβ42—We investigated whether and how Aβ46 is processed by γ-secretase using the Aβ46-accumulating LDM domains. Before incubation, a large amount of Aβ46 was present, and other Aβ species were hardly detectable in the isolated LDM domains (Fig. 2, top panel, 2nd lane from left). Following incubation at 37 °C for 30 min, the Aβ band representing the total Aβ shifted to a band with faster mobility (Fig. 2, top panel, 3rd lane from left). This suggests that Aβ46 is converted to an Aβ species that is shorter than Aβ46. Western blotting with BA27 or BC05 and analysis by SDS/urea gel system indicated that Aβ40, Aβ42, and Aβ43 together with AICD were produced during incubation. These results strongly suggest that both γ- and ϵ-cleavages proceed in parallel in the isolated LDM domains. We next examined the effects of L685,458, a transition state analogue inhibitor of γ-secretase, on the Aβ-producing LDM domains. As expected, L685,458 efficiently suppressed ϵ-cleavage of βCTF (Fig. 2, 2nd panel, 3rd lane from left). In contrast, the production of Aβ40/43 was not suppressed as shown by the conventional Tris/Tricine gel electrophoresis (Fig. 2, top panel, 4th lane from left). Western blotting with BA27 or BC05 and SDS/urea gel electrophoresis confirmed that Aβ40 and Aβ43, but not Aβ42, were produced even in the presence of L685,458. Aβ46 was no longer detectable by either gel system after incubation (Fig. 2, top and bottom panels). These results strongly suggest that Aβ40 and Aβ43 are generated from preaccumulated Aβ46 but not from pre-existing βCTF, in the presence of L685,458, and that Aβ42 does not come from preaccumulated Aβ46. In contrast, incubation in the presence of DAPT led to minimal or no decrease in the level of Aβ46 (Fig. 2, top panel, 3rd lane from right). Instead, a very small amount of AICD and detectable amounts of Aβ40 and Aβ43 were produced. The combination of DAPT and L685,458 suppressed both the processing of Aβ46 (Fig. 2, top panel, 2nd lane from right) and the production of AICD, although trace amounts of Aβ40 and Aβ43 and a very small decrease in the amount of Aβ46 were still observed. DAPT Suppression of Aβ46 Processing—Dose-dependent effects of DAPT on the levels of Aβ produced were assessed. Isolated LDM domains were incubated for 30 min at 37 °C in the presence of 1 μm L685,458 and various concentrations of DAPT (Fig. 3). The presence of L685,458 completely prevented the production of AICD (see the 2nd panel in Fig. 2). Increasing concentrations of DAPT gradually decreased the amounts of Aβ40 and Aβ43 in a similar pattern and increased those of Aβ46 left in the membrane. These reciprocal changes support the above interpretation that both Aβ40 and Aβ43 are processed from Aβ46, and the DAPT suppression of the processing indicates that it is mediated through γ-secretase. These results are consistent with our previous observations that DAPT but not L685,458 induces the intracellular accumulation of Aβ46 (and Aβ43) (10Qi-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 (302) Google Scholar), possibly because DAPT preferentially blocks γ-cleavage, whereas L658,458 preferentially blocks ϵ-cleavage. It is possible that the lesser susceptibility of ϵ-cleavage and greater susceptibility of γ-cleavage to DAPT are presumably responsible for intracellular accumulation of Aβ43 and Aβ46. The distinct dose-dependent profiles for Aβ43 and Aβ46 may result from a small difference in the susceptibility of γ-cleavage to DAPT in producing Aβ43 or Aβ46. Time Course of Aβ46 Processing—To learn more about how Aβ46 is processed to Aβ43 and Aβ40, the time-dependent profiles were examined in the presence of 1 μm L685,458, which completely blocks new recruitment of βCTF (Fig. 4). Because Aβ46 was swiftly processed to Aβ40/43 and rapidly used up at 37 °C (data not shown), the samples were incubated at 25 °C for the times indicated, and the amount of each Aβ at each time point was quantified by quantitative Western blotting. Both Aβ40 and Aβ43 levels increased in a linear fashion for up to 10 min, and this increase was accompanied by an apparently exponential decrease in the amount of Aβ46. For representative numbers, the concentration of Aβ46 in the reaction mixture was 158 fmol/ml at 0 min, and this decreased to 20 fmol/ml at 20 min. Thus, the concentration of Aβ46 decreased by138 fmol/ml during the 20-min incubation. During the incubation, the concentrations of Aβ40 and Aβ43 increased to 70 and 39 fmol/ml, respectively. Thus, the sum of the newly produced Aβ was 109 fmol/ml, which accounts for most of the decrease in Aβ46. We reported previously that the DAPT treatment causes dose-dependent accumulation of longer Aβs, which accompanies a great decrease in Aβ40 within CHO cells co-expressing βCTF and various types of PS1/2 (10Qi-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 (302) Google Scholar, 12Yagishita S. Morishima-Kawashima M. Tanimura Y. Ishiura S. Ihara Y. Biochemistry. 2006; 45: 3952-3960Crossref PubMed Scopus (54) Google Scholar). Although the particular Aβ species that accumulate within the cells vary among the cell lines, a profound decrease in Aβ40 is invariably associated with a small but significant to a large increase in Aβ46, suggesting a close relationship between Aβ40 and Aβ46 production (10Qi-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 (302) Google Scholar, 12Yagishita S. Morishima-Kawashima M. Tanimura Y. Ishiura S. Ihara Y. Biochemistry. 2006; 45: 3952-3960Crossref PubMed Scopus (54) Google Scholar). These results can be explained based on the stepwise (successive) cleavage hypothesis that Aβ49 generated by ϵ-cleavage is processed to Aβ46, and Aβ46 is then processed to Aβ43, and finally, Aβ43 is processed to Aβ40. Consistent with this model, blocking the cleavage at the midportion between γ- and ϵ-sites by a Trp stretch replacement remarkably suppressed the generation of Aβ40/42 (20Sato T. Tanimura Y. Hirotani N. Saido T.C. Morishima-Kawashima M. Ihara Y. FEBS Lett. 2005; 579: 2907-2912Crossref PubMed Scopus (26) Google Scholar). Thus, we established an Aβ-producing system using the Aβ46-accumulating LDM domains. According to our data (15Wada 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) and data from others (19Wahrle S. Das P. Nyborg A.C. McLendon C. Shoji M. Kawarabayashi T. Younkin L.H. Younkin S.G. Golde T.E. Neurobiol. Dis. 2002; 9: 11-23Crossref PubMed Scopus (355) Google Scholar), γ-secretase activity is enriched in LDM domains and accumulating Aβ46 localized predominantly to the LDM domains in DAPT-treated cells (Fig. 1B). The treatment of Aβ46-accumulating LDM domains with L685,458 completely suppressed the production of AICD, but Aβ40 and Aβ43 were still produced, and concomitantly, Aβ46 became undetectable (Fig. 2). Thus, it is plausible that preaccumulated Aβ46 was processed to Aβ43 and Aβ40. The quantification and time-dependent profiles of the produced and converted Aβ levels (Fig. 4) strongly support this explanation. Interestingly, L685,458 failed to block the γ-cleavage, that is the processing from Aβ46 to Aβ43 and Aβ40. This suggests that L685,458 preferentially inhibits ϵ-cleavage rather than γ-cleavage. This is consistent with our previous observation (10Qi-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 (302) Google Scholar) that L685,458 does not induce accumulation of intracellular Aβs. In contrast, treatment with DAPT suppressed the overall processing of Aβ46 and caused leaky production of AICD, suggesting that in contrast to L685,458, DAPT preferentially inhibits γ-cleavage. These observations are consistent with those by Zhao et al. (21Zhao G. Cui M.Z. Mao G. Dong Y. Tan J. Sun L. Xu X. J. Biol. Chem. 2005; 280: 37689-37697Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar) that L685,458 specifically inhibits ϵ-cleavage and ζ-cleavage (cleavage between Ile-45 and Val-46), whereas DAPT preferentially inhibits γ-cleavage. Differential sensitivity to L685,458 and DAPT may be explained by differences in their γ-secretase-binding sites. L685,458 is a transition state analogue and directly targets the catalytic residue Asp on the transmembrane domains 6 and 7 of PS1 or 2 (22Li 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 (857) Google Scholar), whereas DAPT is a nontransition state analogue and does not target these residues (23Morohashi Y. Kan T. Tominari Y. Fuwa H. Okamura Y. Watanabe N. Sato C. Natsugari H. Fukuyama T. Iwatsubo T. Tomita T. J. Biol. Chem. 2006; 281: 14670-14676Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 24Sato C. Morohashi Y. Tomita T. Iwatsubo T. J. Neurosci. 2006; 26: 12081-12088Crossref PubMed Scopus (131) Google Scholar). Because Aβ46 is likely an intermediate product, accumulating Aβ46 may remain to occupy the catalytic pore of γ-secretase and prevent L685,458 from entering the catalytic site. Thus, Aβ46 that occupies the catalytic site could be processed by γ-secretase despite the presence of L685,458. The association between accumulating Aβ46 and PS1 was shown by co-immunoprecipitation in compound E-treated cells (21Zhao G. Cui M.Z. Mao G. Dong Y. Tan J. Sun L. Xu X. J. Biol. Chem. 2005; 280: 37689-37697Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). These explain our observation that L685,458 failed to suppress Aβ46 processing. Moreover, the apparent suppression of ϵ-cleavage by L685,458 may be caused partly by pre-existing Aβ46, which prevents the entry of βCTF into the catalytic pore of γ-secretase. On the other hand, DAPT suppresses γ-secretase activity by binding to a different site from the catalytic site. Thus, pre-existing Aβ46 may not prevent the binding of DAPT to γ-secretase. Our results also provide a strong support to the notion that the Aβ40-producing process is separate from the Aβ42-producing process. The latter process was less sensitive to L685,458 than was the former. Our preliminary observations indicate that the accumulating Aβ45 in the LDM domains obtained from N141I mtPS2-transfected cells is not processed to Aβ42 by incubation, suggesting that both processes are functionally different from each other. Furthermore, the CHAPSO-solubilized reconstituted system for γ-secretase gave unexpected results. Aβ46, a putative precursor of Aβ40 and Aβ43, is undetectable, whereas Aβ45 is clearly detectable (25Kakuda 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). This again points to the possibility that the Aβ42-producing process is separate from the Aβ40-producing process. However, Zhao et al. (26Zhao G. Tan J. Mao G. Cui M.Z. Xu X. J. Neurochem. 2007; 100: 1234-1246Crossref PubMed Scopus (39) Google Scholar) recently reported that Aβ46 is an intermediate precursor of both Aβ40 and Aβ42. Inconsistency between their results and ours may have arisen from the differences in the assay systems for γ-secretase; Zhao et al. (26Zhao G. Tan J. Mao G. Cui M.Z. Xu X. J. Neurochem. 2007; 100: 1234-1246Crossref PubMed Scopus (39) Google Scholar) used either cell culture or a cell-free system using the total membrane fraction, whereas we used a cell-free system using the LDM domains. Most importantly, the pH (7.0) of our cell-free system differed from that of the system used by Zhao et al. (26Zhao G. Tan J. Mao G. Cui M.Z. Xu X. J. Neurochem. 2007; 100: 1234-1246Crossref PubMed Scopus (39) Google Scholar) (pH 6.4). We found that Aβ processing at lower pH (pH 6.5) proceeds in a γ-secretase-independent manner by another protease, presumably involving cathepsin D, whose activity localized to fraction 4. 4S. Yagishita, M. Morishima-Kawashima, and Y. Ihara, unpublished observations. Thus, isolation of the LDM domains in our study may have successfully removed contaminating protease activities that are associated with the total membrane fraction. Two models could explain the mechanism of Aβ46 processing observed here. One is that Aβ46 is first processed to Aβ43, which is then processed to Aβ40. This is referred to here as the successive reaction model. The other mechanism is that Aβ40 and Aβ43 are simultaneously produced from Aβ46. This is referred to here as the nonsuccessive model. To determine which model is more plausible, we carefully examined the time-dependent profiles of the processing in the presence of L685,458 at 25 °C (see Fig. 4), because incubation at 25 °C made the reaction slow enough to accurately follow the production rates. The following successive reactions assume that these first-order reactions are catalyzed by the same γ-secretase: Aβ46 → Aβ43 → Aβ40. The concentration of the intermediate (Aβ43) is given by the following: [Aβ43]t = [Aβ46]0k1/k2 – k1 (exp(–k1t) – exp(–k2t)), where k1 and k2 are rate constants for the first and second reactions. The successive reactions differ from simple reactions and have unusual characteristics. The concentration extremum is detectable with an intermediate product. In this case, Aβ43 would reach a maximal concentration at t = (ln(k1/k2))/k1 – k2. Regarding Aβ40, the initial rate is zero, and an induction period is present. However, even careful observations did not enable us to detect such unusual characteristics. This suggests that processing of Aβ46 does not undergo successive reactions but that Aβ40 and Aβ43 are produced directly from Aβ46, presumably by releasing hexapeptide, IATVIV, and tripeptide, VIV, respectively. This kind of cleavage still follows the α-helical model for γ-cleavage (10Qi-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 (302) Google Scholar). Finally, we note that it remains unknown whether the nonsuccessive model proposed from our study on the LDM domains is applicable to the original cell-free system. In our experience, the CHAPSO-solubilized, reconstituted system appears to follow a nonsuccessive model, because the possible intermediate products did not achieve a maximal concentration during the time course of the study (25Kakuda 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). Thus, various sized Aβs may have been produced independently from the ϵ-cleaved products, Aβ48 and Aβ49, apparently in a nonsuccessive manner. It is possible that the presence of an intact membrane and some as-yet unidentified components provide the properties of successive reactions originally postulated. We thank Takeda Chemical Industries for BA27 and BC05. Download .pdf (.26 MB) Help with pdf files" @default.
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