The Mechanism of γ-Secretase Activities through High Molecular Weight Complex Formation of Presenilins Is Conserved inDrosophila melanogaster and Mammals
2002; Elsevier BV; Volume: 277; Issue: 51 Linguagem: Inglês
10.1074/jbc.m205352200
ISSN1083-351X
AutoresNobumasa Takasugi, Yasuko Takahashi, Yuichi Morohashi, Taisuke Tomita, Takeshi Iwatsubo,
Tópico(s)Supramolecular Self-Assembly in Materials
ResumoMutations in presenilin 1 (PS1) and PS2 genes contribute to the pathogenesis of early onset familial Alzheimer's disease by increasing secretion of the pathologically relevant Aβ42 polypeptides. PS genes are also implicated in Notch signaling through proteolytic processing of the Notch receptor in Caenorhabditis elegans, Drosophila melanogaster, and mammals. Here we show thatDrosophila PS (Psn) protein undergoes endoproteolytic cleavage and forms a stable high molecular weight (HMW) complex inDrosophila S2 or mouse neuro2a (N2a) cells in a similar manner to mammalian PS. The loss-of-function recessive point mutations located in the C-terminal region of Psn, that cause an early pupal-lethal phenotype resembling Notch mutant in vivo, disrupted the HMW complex formation, and abolished γ-secretase activities in cultured cells. The overexpression of Psn in mouse embryonic fibroblasts lacking PS1 andPS2 genes rescued the Notch processing. Moreover, disruption of the expression of Psn by double-stranded RNA-mediated interference completely abolished the γ-secretase activity in S2 cells. Surprisingly, γ-secretase activity dependent on wild-type Psn was associated with a drastic overproduction of Aβ1–42 from human βAPP in N2a cells, but not in S2 cells. Our data suggest that the mechanism of γ-secretase activities through formation of HMW PS complex, as well as its abolition by loss-of-function mutations located in the C terminus, are highly conserved features inDrosophila and mammals. Mutations in presenilin 1 (PS1) and PS2 genes contribute to the pathogenesis of early onset familial Alzheimer's disease by increasing secretion of the pathologically relevant Aβ42 polypeptides. PS genes are also implicated in Notch signaling through proteolytic processing of the Notch receptor in Caenorhabditis elegans, Drosophila melanogaster, and mammals. Here we show thatDrosophila PS (Psn) protein undergoes endoproteolytic cleavage and forms a stable high molecular weight (HMW) complex inDrosophila S2 or mouse neuro2a (N2a) cells in a similar manner to mammalian PS. The loss-of-function recessive point mutations located in the C-terminal region of Psn, that cause an early pupal-lethal phenotype resembling Notch mutant in vivo, disrupted the HMW complex formation, and abolished γ-secretase activities in cultured cells. The overexpression of Psn in mouse embryonic fibroblasts lacking PS1 andPS2 genes rescued the Notch processing. Moreover, disruption of the expression of Psn by double-stranded RNA-mediated interference completely abolished the γ-secretase activity in S2 cells. Surprisingly, γ-secretase activity dependent on wild-type Psn was associated with a drastic overproduction of Aβ1–42 from human βAPP in N2a cells, but not in S2 cells. Our data suggest that the mechanism of γ-secretase activities through formation of HMW PS complex, as well as its abolition by loss-of-function mutations located in the C terminus, are highly conserved features inDrosophila and mammals. Mutations in presenilin (PS1) 1The abbreviations used are: PS1, presenilin 1; AD, Alzheimer's disease; Aβ, amyloid β peptide; βAPP, β-amyloid precursor protein; CHAPSO, 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate; CHX, cycloheximide; CTF, C-terminal fragment; dsRNA, double-stranded RNA; EGFP, enhanced green fluorescent protein; ELISA, enzyme-linked immunosorbent assay; FAD, familial Alzheimer's disease; FL, full-length; N2a, mouse neuro2a neuroblastoma; NICD, Notch intracellular domain; NTF, N-terminal fragment; RNAi, double-stranded RNA-mediated interference; TGN, trans-Golgi network; TMD, transmembrane domain; HMW, high molecular weight; LMW, low molecular weight orPS2 genes account for the majority of early-onset familial Alzheimer's disease (FAD), and these mutations cause an increase in the ratio or levels of production of amyloid β peptides ending at position 42 (Aβ42), that most readily form amyloid deposits (1Selkoe D.J. Physiol. Rev. 2001; 81: 741-766Crossref PubMed Scopus (5168) Google Scholar). Presenilins are polytopic integral membrane proteins that span the membrane eight times and undergo endoproteolysis (2Thinakaran G. Borchelt D.R. Lee M.K. Slunt H.H. Spitzer L. Kim G. Ratovitski T. Davenport F. Nordstedt C. Seeger M. Hardy J. Levey A.I. Gandy S.E. Jenkins N.A. Copeland N.G. Price D.L. Sisodia S.S. Neuron. 1996; 17: 181-190Abstract Full Text Full Text PDF PubMed Scopus (940) Google Scholar). The endoproteolytic fragments of PS are incorporated into a high molecular weight (HMW) complex (3Seeger M. Nordstedt C. Petanceska S. Kovacs D.M. Gouras G.K. Hahne S. Fraser P. Levesque L. Czernik A.J. St. George Hyslop P.S. Sisodia S.S. Thinakaran G. Tanzi R.E. Greengard P. Gandy S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5090-5094Crossref PubMed Scopus (133) Google Scholar, 4Yu G. Chen F. Levesque G. Nishimura M. Zhang D.M. Levesque L. Rogaeva E. Xu D. Liang Y. Duthie M. St George-Hyslop P.H. Fraser P.E. J. Biol. Chem. 1998; 273: 16470-16475Abstract Full Text Full Text PDF PubMed Scopus (366) Google Scholar) and are highly stabilized (t12 = ∼20 h), whereas holoprotein is rapidly degraded (t12 = ∼2 h) (5Ratovitski T. Slunt H.H. Thinakaran G. Price D.L. Sisodia S.S. Borchelt D.R. J. Biol. Chem. 1997; 272: 24536-24541Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). PS is implicated in γ-cleavage of βAPP, the final step in the generation of Aβ peptides, as well as in the γ-cleavage-like intramembranous proteolysis of various transmembrane proteins (e.g. Notch, ErbB4, E-cadherin, and LRP) (reviewed in Ref.6Fortini M.E. Nat. Rev. Mol. Cell. Biol. 2002; 3: 673-684Crossref PubMed Scopus (343) Google Scholar). Although the precise role of PS in the intramembranous proteolysis still remains unknown, following lines of evidence suggest that PS is a catalytic component of γ-secretase. First, the ablation of PS genes in mice inactivated the total γ-secretase activities (7Herreman A. Serneels L. Annaert W. Collen D. Schoonjans L. De Strooper B. Nat. Cell Biol. 2000; 2: 461-462Crossref PubMed Scopus (450) Google Scholar, 8Zhang Z. Nadeau P. Song W. Donoviel D. Yuan M. Bernstein A. Yankner B.A. Nat. Cell Biol. 2000; 2: 463-465Crossref PubMed Scopus (359) Google Scholar). Second, mutating either of the two conserved aspartate residues within the transmembrane domains (TMD) 6 and 7 of PS inhibited the γ-secretase activities (9Wolfe M.S. Xia W. Ostaszewski B.L. Diehl T.S. Kimberly W.T. Selkoe D.J. Nature. 1999; 398: 513-517Crossref PubMed Scopus (1692) Google Scholar). Third, the γ-secretase activity solubilized by a mild detergent, CHAPSO, was immunoprecipitated by antibodies against PS1 in HMW fractions (10Li 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 (499) Google Scholar). Lastly, the transition-state analogue γ-secretase inhibitors that are conjugated with photoaffinity labeling and/or biotin tags directly labeled the PS fragments (11Li 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 (865) Google Scholar, 12Esler W.P. Kimberly W.T. Ostaszewski B.L. Diehl T.S. Moore C.L. Tsai J.Y. Rahmati T. Xia W. Selkoe D.J. Wolfe M.S. Nat. Cell Biol. 2000; 2: 428-434Crossref PubMed Scopus (506) Google Scholar, 13Seiffert D. Bradley J.D. Rominger C.M. Rominger D.H. Yang F. Meredith J.E. Wang Q. Roach A.H. Thompson L.A. Spitz S.M. Higaki J.N. Prakash S.R. Combs A.P. Copeland R.A. Arneric S.P. Hartig P.R. Robertson D.W. Cordell B. Stern A.M. Olson R.E. Zaczek R. J. Biol. Chem. 2000; 275: 34086-34091Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar). Recently, functional γ-secretase complex containing PS fragments was partially purified by an immobilized γ-secretase inhibitor (14Esler 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 (344) Google Scholar). Taken together, it is strongly suggested that the stabilized HMW complex of PS represents the functional form of γ-secretase and that the PS fragments harbor the catalytic center of γ-secretase. PS is an evolutionarily conserved protein that is present in every multicellular organism including vertebrates and invertebrates as well as plants, and the primary amino acid sequences of the C-terminal region of PS are highly conserved (15Tomita T. Watabiki T. Takikawa R. Morohashi Y. Takasugi N. Kopan R. De Strooper B. Iwatsubo T. J. Biol. Chem. 2001; 276: 33273-33281Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). We have previously shown that stabilization and formation of the HMW PS complex that are dependent on the integrity of the PS C terminus is required for the γ-secretase activity (16Tomita T. Takikawa R. Koyama A. Morohashi Y. Takasugi N. Saido T.C. Maruyama K. Iwatsubo T. J. Neurosci. 1999; 19: 10627-10634Crossref PubMed Google Scholar). Missense mutations that replace the 1st proline of the C-terminal PALP motif, which is completely conserved in all PS family members, with leucine, lead to a loss-of-function of PS inDrosophila melanogaster presenilin (Psn) as well as in Caenorhabditis elegans Spe-4 (17Arduengo P.M. Appleberry O.K. Chuang P. L'Hernault S.W. J. Cell Sci. 1998; 111: 3645-3654Crossref PubMed Google Scholar, 18Guo Y. Livne-Bar I. Zhou L. Boulianne G.L. J. Neurosci. 1999; 19: 8435-8442Crossref PubMed Google Scholar). Moreover,PsnB3 allele, another loss-of-function mutant ofPsn, that results in an amino acid substitution (G516E) in the C terminus of Psn has been reported (19Lukinova N.I. Roussakova V.V. Fortini M.E. Genetics. 1999; 153: 1789-1797PubMed Google Scholar). This glycine residue also is conserved in almost all known PS family members except for the C. elegans Hop-1 protein. We have previously shown that the 1st proline of the PALP motif is required for the stabilization, complex formation, and γ-secretase activities of human PS in mammalian cells (15Tomita T. Watabiki T. Takikawa R. Morohashi Y. Takasugi N. Kopan R. De Strooper B. Iwatsubo T. J. Biol. Chem. 2001; 276: 33273-33281Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). However, the effects of the loss-of-function mutations (P507L or G516E) on the metabolism and complex formation of Psn polypeptides still remain unknown. In this study, we examined the modes of processing, complex formation, and function of Psn protein in Drosophila S2 cells as well as in mammalian cells and compared them with those of human PS. Full-length (FL) cDNAs encoding wild type, FAD mutant (N141I) human PS2 in pcDNA3 (Invitrogen, Carlsbad, CA) were obtained as described (20Tomita T. Maruyama K. Saido T.C. Kume H. Shinozaki K. Tokuhiro S. Capell A. Walter J. Grunberg J. Haass C. Iwatsubo T. Obata K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2025-2030Crossref PubMed Scopus (353) Google Scholar). A full-length cDNA encoding 508 amino acid residues of full-lengthPsn in pOT2 vector was provided by Dr. G. L. Boulianne (21Boulianne G.L. Livne-Bar I. Humphreys J.M. Liang Y. Lin C. Rogaev E. St. George-Hyslop P. Neuroreport. 1997; 8: 1025-1029Crossref PubMed Scopus (63) Google Scholar). A cDNA coding for the Psn open reading frame was generated by PCR using PfuTurbo (Stratagene, La Jolla, CA), and the following oligonucleotides were used as a PCR primer: 5′-GAATTCATGGCTGCTGTCAATCTCCAG-3′ as a forward primer and 5′-GGGCTCGAGTTATATAAACACCTGCTT-3′ as a reverse primer. The amplified cDNA was subcloned into pcDNA3 or pAc5.1/V5-His A vector (Invitrogen). A cDNA encoding enhanced green fluorescent protein (EGFP) was digested from pEGFP-N1 (BD Biosciences Clontech, Palo Alto, CA) and subcloned into pAc5.1/V5-His A vector. cDNAs encoding Psn/D461A, Psn/P507L, Psn/G516E, (amino acid numbering based on Psn541) or human PS2/G423E were generated by the long-PCR protocol (15Tomita T. Watabiki T. Takikawa R. Morohashi Y. Takasugi N. Kopan R. De Strooper B. Iwatsubo T. J. Biol. Chem. 2001; 276: 33273-33281Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 22Morohashi Y. Hatano N. Ohya S. Takikawa R. Watabiki T. Takasugi N. Imaizumi Y. Tomita T. Iwatsubo T. J. Biol. Chem. 2002; 277: 14965-14975Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar) using cDNAs encoding Psn, wild type, or mutant PS2 in pcDNA3 vector as the templates, using the following primer pairs: 5′-GGCCTCGGCGCATTCATCTTCTACTCGGTACTAGTGGGC-3′ for Psn/D461A, 5′-GGGCAGGGCTAGCAGCGCCTTGCGCCAAATGGC-3′ for Psn/P507L, 5′-GGCGAAGCAAAATATGAGCTCGAACGTTATTGAGATGGGCAGGGC-3′ for Psn/G516E, 5′-GTAAAAGATGAGCTCGAACGTGATGGAGATGGGGAG-3′ for PS2/G423E as forward primers, 5′-GCCCACTAGTACCGAGTAGAAGATGAATGCGCCGAGGCC-3′ for Psn/D461A, 5′-GCCATTTGGCGCAAGGCGCTGCTAGCCCTGCCC-3′ for Psn/P507L, 5′-GCCCTGCCCATCTCAATAACGTTCGAGCTCATATTTTGCTTCGCC-3′ for Psn/G516E, 5′-CTCCCCATCTCCATCACGTTCGAGCTCATCTTTTAC-3′ for PS2/G423E as reverse primers, respectively. Schematic depictions of modified PS derivatives used in this study are shown in Fig. 1. A cDNA encoding the C-terminal 99 amino acids of βAPP fused to a signal peptide of rat preproenkephalin cDNA (SC100) was generated by PCR using a SC100 cDNA in pcDNA3.1/Hygro(+) vector (Invitrogen) as a template, using the following primer pairs: 5′-GGTACCACCATGGCGCAGTTCCTG-3′ as a forward primer and 5′-GAGCAGATGCAGAACTAGCTCGAG-3′ as a reverse primer and subcloned into pAc5.1/V5-His A vector (20Tomita T. Maruyama K. Saido T.C. Kume H. Shinozaki K. Tokuhiro S. Capell A. Walter J. Grunberg J. Haass C. Iwatsubo T. Obata K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2025-2030Crossref PubMed Scopus (353) Google Scholar, 23Iwata H. Tomita T. Maruyama K. Iwatsubo T. J. Biol. Chem. 2001; 276: 21678-21685Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). A cDNA encoding SC100/I716F was generated by the long-PCR protocol using cDNAs encoding SC100 in pAc5.1/V5-His A vector as templates, using following primer pairs: 5′-GTCATAGCGACAGTGTTCGTCATCACCTTGG-3′ as forward primers, 5′-CCAAGGTGATGACGAACACTGTCGCTATGAC-3′ as reverse primers. All constructs were sequenced using Thermosequenase (Amersham Biosciences) on an automated sequencer (Li-Cor, Lincolin, NE). cDNAs encoding mouse NotchΔE in pCS2+MT vector and βAPP695 carrying a Swedish mutation (βAPPNL) in pCEP4 (Invitrogen) have been described previously (23Iwata H. Tomita T. Maruyama K. Iwatsubo T. J. Biol. Chem. 2001; 276: 21678-21685Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 24Schroeter E.H. Kisslinger J.A. Kopan R. Nature. 1998; 393: 382-386Crossref PubMed Scopus (1362) Google Scholar). Mouse neuro2a (N2a) neuroblastoma cells and SV40-transformed mouse embryonic fibroblasts (MEF) derived from PS1−/−PS2−/− littermates (provided by Dr. B. De Strooper) were maintained as described (15Tomita T. Watabiki T. Takikawa R. Morohashi Y. Takasugi N. Kopan R. De Strooper B. Iwatsubo T. J. Biol. Chem. 2001; 276: 33273-33281Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Generation of stable N2a cell lines co-expressing βAPPNLand NotchΔE (NL/N) were described previously (25Tomita T. Katayama R. Takikawa R. Iwatsubo T. FEBS Lett. 2002; 520: 117-121Crossref PubMed Scopus (63) Google Scholar). Stable N2a NL/N cell lines expressing Psn or PS2 derivatives were generated by transfecting cDNAs using LipofectAMINE and selected in Dulbecco's modified Eagle's medium containing both hygromycin (Wako) at 250 μg/ml and G418 (Calbiochem, San Diego, CA) at 500 μg/ml. Transient transfection of cDNAs into MEF cells were performed using LipofectAMINE 2000 (Invitrogen) according to the manufacturer's instructions. After 24 h of transfection, 10 mmbutyric acid was added for 24 h to drive protein expression. Drosophila Schneider (S2) cells were maintained in Schneider's insect medium (Sigma) supplemented with 10% fetal bovine serum, 5% peptone, and penicillin/streptomycin (S2 medium) at 24 °C (26Hirasaka S. Kanuka H. Shoji S. Yoshikawa S. Okano H. Miura M. J. Cell Sci. 1998; 111: 667-673PubMed Google Scholar). Transient transfection of cDNAs into S2 cells was performed using Cellfectin (Invitrogen) according to the manufacturer's instructions, and samples were collected after 48 h of transfection. Stable S2 cell lines were generated by transfection of cDNAs in pAc5.1/V5-His A vector together with those in pCoHygro (Invitrogen) vector (ratio of transfected cDNAs; 2:0.1 μg) using Cellfectin and selection in S2 medium containing hygromycin at 250 μg/ml. For the production of the double-stranded RNA (dsRNA), transcription templates that contained T7 RNA promoter sequences on each end were generated by PCR using the following oligonucleotides containing the T7 RNA polymerase binding site as primer pairs: 5′-TTAATACGACTCACTATAGGGAGAATGGCTGCTGTCAAT-3′ for Psn, 5′-TTAATACGACTCACTATAGGGAGAATGGTGAGCAAGGGC-3′ for EGFP as sense primers, 5′-TTAATACGACTCACTATAGGGAGAGACATCATTCCGACC-3′ for Psn, 5′-TTAATACGACTCACTATAGGGAGATTACTTGTACAGCTC-3′ for EGFP as reverse primers, respectively. dsRNAs were prepared from transcription templates by using MEGAscript T7 KIT (Ambion, Austin, TX) and transfected into S2 cells using Cellfectin. Cell lysates and conditioned media were harvested after incubation for indicated times. The following rabbit polyclonal antibodies were generated and used: anti-GDN1 against glutathione S-transferase (GST) fused to amino acids 2–52 of Psn, anti-GDL1 against GST fused to amino acids 358–426 of Psn; anti-G2L against GST fused to amino acids 301–361 of human PS2, anti-G2N4 against GST fused to amino acids 2–59 of human PS2, anti-G1Nr2 against GST fused to amino acids 2–70 of human PS1, and anti-G1L3 against GST fused to amino acids 297–379 of human PS1 have been previously described (15Tomita T. Watabiki T. Takikawa R. Morohashi Y. Takasugi N. Kopan R. De Strooper B. Iwatsubo T. J. Biol. Chem. 2001; 276: 33273-33281Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 16Tomita T. Takikawa R. Koyama A. Morohashi Y. Takasugi N. Saido T.C. Maruyama K. Iwatsubo T. J. Neurosci. 1999; 19: 10627-10634Crossref PubMed Google Scholar, 20Tomita T. Maruyama K. Saido T.C. Kume H. Shinozaki K. Tokuhiro S. Capell A. Walter J. Grunberg J. Haass C. Iwatsubo T. Obata K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2025-2030Crossref PubMed Scopus (353) Google Scholar, 22Morohashi Y. Hatano N. Ohya S. Takikawa R. Watabiki T. Takasugi N. Imaizumi Y. Tomita T. Iwatsubo T. J. Biol. Chem. 2002; 277: 14965-14975Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 27Tomita T. Tokuhiro S. Hashimoto T. Aiba K. Saido T.C. Maruyama K. Iwatsubo T. J. Biol. Chem. 1998; 273: 21153-21160Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). The rabbit polyclonal antibody C4 against the cytoplasmic C terminus of human βAPP was kindly provided by Dr. Y. Ihara (University of Tokyo). The mouse monoclonal antibodies were purchased from Stressgen (anti-KDEL), Transduction Laboratory (anti-Adaptin-γ), and Roche Diagnostics (anti-c-Myc (9E10)), respectively. Preparation of cell lysates, immunoblot analysis, cycloheximide treatment, glycerol velocity centrifugation, and subcellular fractionation using Iodixanol gradient centrifugation were performed as previously described (15Tomita T. Watabiki T. Takikawa R. Morohashi Y. Takasugi N. Kopan R. De Strooper B. Iwatsubo T. J. Biol. Chem. 2001; 276: 33273-33281Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 22Morohashi Y. Hatano N. Ohya S. Takikawa R. Watabiki T. Takasugi N. Imaizumi Y. Tomita T. Iwatsubo T. J. Biol. Chem. 2002; 277: 14965-14975Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar,23Iwata H. Tomita T. Maruyama K. Iwatsubo T. J. Biol. Chem. 2001; 276: 21678-21685Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Two site ELISAs that specifically detect the C terminus of Aβ were used as described. BAN50 is a monoclonal antibody raised against a synthetic peptide of human Aβ1–16; it preferentially reacts with the N-terminal portion of human Aβ starting at Asp-1, but does not cross-react with N-terminally truncated Aβ nor with rodent-type Aβ (20Tomita T. Maruyama K. Saido T.C. Kume H. Shinozaki K. Tokuhiro S. Capell A. Walter J. Grunberg J. Haass C. Iwatsubo T. Obata K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2025-2030Crossref PubMed Scopus (353) Google Scholar, 28Asami-Odaka A. Ishibashi Y. Kikuchi T. Kitada C. Suzuki N. Biochemistry. 1995; 34: 10272-10278Crossref PubMed Scopus (150) Google Scholar). BA27 and BC05 that specifically recognize the C terminus of Aβ40 and Aβ42, respectively, were conjugated with horseradish peroxidase and used as detector antibodies. Culture media were collected after an appropriate incubation period and subjected to BAN50/BA27 or BAN50/BC05 ELISAs as described (20Tomita T. Maruyama K. Saido T.C. Kume H. Shinozaki K. Tokuhiro S. Capell A. Walter J. Grunberg J. Haass C. Iwatsubo T. Obata K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2025-2030Crossref PubMed Scopus (353) Google Scholar, 29Iwatsubo T. Odaka A. Suzuki N. Mizusawa H. Nukina N. Ihara Y. Neuron. 1994; 13: 45-53Abstract Full Text PDF PubMed Scopus (1558) Google Scholar). Drosophila presenilin (Psn) gene encodes 508–541 amino acid proteins with ∼50% identity to its vertebrate counterparts (21Boulianne G.L. Livne-Bar I. Humphreys J.M. Liang Y. Lin C. Rogaev E. St. George-Hyslop P. Neuroreport. 1997; 8: 1025-1029Crossref PubMed Scopus (63) Google Scholar). The occurrence of endoproteolytic cleavage of Psn protein in vivo and in theDrosophila S2 cell line has also been documented, although a detailed analysis on the metabolism of Psn polypeptides is yet to be performed (18Guo Y. Livne-Bar I. Zhou L. Boulianne G.L. J. Neurosci. 1999; 19: 8435-8442Crossref PubMed Google Scholar, 30Nowotny P. Gorski S.M. Han S.W. Philips K. Ray W.J. Nowotny V. Jones C.J. Clark R.F. Cagan R.L. Goate A.M. Mol. Cell Neurosci. 2000; 15: 88-98Crossref PubMed Scopus (33) Google Scholar). To examine the expression and metabolism of endogenous and transfected Psn proteins in S2 or mouse N2a cell lines, we stably transfected these cells with Psn and analyzed by immunoblotting with antibodies against the N terminus or hydrophilic 6th loop of Psn (i.e. anti-GDN1 and anti-GDL1, respectively). Immunoblot analysis of lysates of untransfected S2 cells revealed a ∼27-kDa N-terminal fragment (NTF) as well as a ∼32-kDa C-terminal fragment (CTF) (Fig.2 A). These bands disappeared when the blots were probed by antibodies preadsorbed with immunogen proteins (data not shown). A faint band of ∼55–60 kDa, corresponding to the full-length Psn protein, also was detectable. These results confirmed the previous reports on the endoproteolysis of Psn as well as the predominance of fragment forms as endogenous Psn, which was similar to those seen with mammalian PS (18Guo Y. Livne-Bar I. Zhou L. Boulianne G.L. J. Neurosci. 1999; 19: 8435-8442Crossref PubMed Google Scholar, 30Nowotny P. Gorski S.M. Han S.W. Philips K. Ray W.J. Nowotny V. Jones C.J. Clark R.F. Cagan R.L. Goate A.M. Mol. Cell Neurosci. 2000; 15: 88-98Crossref PubMed Scopus (33) Google Scholar). We next analyzed the lysates of S2 cells stably transfected with Psn (Fig. 2 A). A ∼55-kDa band corresponding to a FL Psn polypeptide was detected by the N- and C-terminal antibodies, whereas the levels of NTF and CTF did not increase, suggesting that the levels of Psn fragments also are regulated by a "limiting co-factor" in a similar manner to mammalian PS (31Thinakaran G. Harris C.L. Ratovitski T. Davenport F. Slunt H.H. Price D.L. Borchelt D.R. Sisodia S.S. J. Biol. Chem. 1997; 272: 28415-28422Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar). To further characterize the metabolism and function of Psn, we stably transfected the Psn cDNA into a mouse N2a cell line stably expressing both βAPPNLand NotchΔE (N2a NL/N cell line) (Fig. 2 A). Immunoblot analysis revealed that Psn polypeptides expressed in N2a NL/N cells underwent endoproteolysis to give rise to NTF and CTF of the same molecular weights as the endogenous ones in S2 cells. Moreover, the overexpression of Psn in N2a NL/N cells compromised the accumulation of endogenous murine PS fragments, suggesting that Psn retains the capacity to replace the endogenous PS by competing for limiting cofactor(s) in a similar fashion to that observed with mammalian PS (Fig. 2 B). Fragments of mammalian PS are highly stabilized and incorporated into HMW protein complexes of ∼200–600 kDa that are distributed in the ER as well as in Golgi/TGN, whereas holoproteins are rapidly degraded, fractionated in the low molecular weight (LMW) range of ∼100–200 kDa, and exclusively distributed in ER (15Tomita T. Watabiki T. Takikawa R. Morohashi Y. Takasugi N. Kopan R. De Strooper B. Iwatsubo T. J. Biol. Chem. 2001; 276: 33273-33281Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). To examine the stability of Psn protein, we treated S2 or N2a NL/N cells stably expressing Psn with cycloheximide (CHX) (Fig.3 A). The levels of endoproteolytic fragments of Psn did not decrease during CHX treatment of 10–12 h, whereas the Psn holoproteins were rapidly degraded similarly to mammalian PS holoproteins. To examine the capacity of Psn proteins to form HMW complexes, we solubilized the membrane fractions of S2 or N2a NL/N cells in 1% CHAPSO, and separated the extracted proteins on a linear glycerol velocity gradient (Fig. 3 B). Endoproteolytic fragments derived from Psn were predominantly distributed in the HMW range of 232–443 kDa, whereas Psn holoproteins were fractionated in the LMW range of 140–232 kDa. Moreover, subcellular fractionation studies using discontinuous Iodixanol gradients showed that endoproteolytic Psn fragments were recovered in fractions containing ER vesicles as well as Golgi membranes, whereas holoproteins were detected in ER fractions in N2a NL/N cells (Fig.3 C and data not shown). These data suggest that Psn proteins are metabolized in Drosophila S2 cells by a similar cellular machinery to that working in mammalian cells, and appropriately metabolized by a mammalian PS-metabolic pathway (i.e.properly folded, assembled with binding partners, stabilized, and forming HMW complex) in mouse N2a cells. The formation of the stabilized HMW complex of mammalian PS, that requires the integrity of the conserved PS C terminus, is essential to the acquisition of γ-secretase activity, and an aspartate residue within 7th TMD (TMD7) is crucial to the γ-secretase activity in mammalian PS (9Wolfe M.S. Xia W. Ostaszewski B.L. Diehl T.S. Kimberly W.T. Selkoe D.J. Nature. 1999; 398: 513-517Crossref PubMed Scopus (1692) Google Scholar, 32Steiner H. Duff K. Capell A. Romig H. Grim M.G. Lincoln S. Hardy J. Yu X. Picciano M. Fechteler K. Citron M. Kopan R. Pesold B. Keck S. Baader M. Tomita T. Iwatsubo T. Baumeister R. Haass C. J. Biol. Chem. 1999; 274: 28669-28673Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar). To verify the effects of missense mutations in Psn that cause Notch (i.e. loss-of-function) phenotype in Drosophila in vivo, on the metabolism of Psn polypeptides, we introduced the two types of amino acid substitutions (i.e. P507L or G516E) and stably expressed the mutant Psn in N2a NL/N cells. In addition, we established N2a NL/N cells stably coexpressing Psn carrying D461A mutation that replaces the highly conserved aspartate residue in the TMD7 with alanine, to see if it works as a dominant negative mutant on γ-cleavage as in mammalian PS (9Wolfe M.S. Xia W. Ostaszewski B.L. Diehl T.S. Kimberly W.T. Selkoe D.J. Nature. 1999; 398: 513-517Crossref PubMed Scopus (1692) Google Scholar, 15Tomita T. Watabiki T. Takikawa R. Morohashi Y. Takasugi N. Kopan R. De Strooper B. Iwatsubo T. J. Biol. Chem. 2001; 276: 33273-33281Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 32Steiner H. Duff K. Capell A. Romig H. Grim M.G. Lincoln S. Hardy J. Yu X. Picciano M. Fechteler K. Citron M. Kopan R. Pesold B. Keck S. Baader M. Tomita T. Iwatsubo T. Baumeister R. Haass C. J. Biol. Chem. 1999; 274: 28669-28673Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar). Structures of the Psn derivatives used here are schematically shown in Fig. 1. Immunoblot analysis of cell lysates showed that neither Psn/P507L, Psn/G516E nor Psn/D461A underwent endoproteolysis to give rise to NTF and CTF that normally occurs with wild type Psn (Fig.4 A). The replacement of endogenous PS1 did not occur in N2a NL/N cells coexpressing Psn/P507L or Psn/G516E. Upon CHX treatment of the N2a cells, the Psn/P507L or Psn/G516E holoproteins were rapidly degraded in a similar manner to wild type Psn holoprotein (Fig. 4 B and data not shown). In contrast, the overexpression of Psn/D461A resulted in a complete replacement of endogenous murine PS1 fragments, and a portion of Psn/D461A was stabilized as a holoprotein, as previously described in aspartate mutants of mammalian PS (i.e. PS1/D385A, PS2/D366A) (9Wolfe M.S. Xia W. Ostaszewski B.L. Diehl T.S. Kimberly W.T. Selkoe D.J. Nature. 1999; 398: 513-517Crossref PubMed Scopus (1692) Google Scholar, 15Tomita T. Watabiki T. Takikawa R. Morohashi Y. Takasugi N. Kopan R. De Strooper B. Iwatsubo T. J. Biol. Chem. 2001; 276: 33273-33281Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 32Steiner H. Duff K. Capell A. Romig H. Grim M.G. Lincoln S. Hardy J. Yu X. Picciano M. Fechteler K. Citron M. Kopan R. Pesold B. Keck S. Baader M. Tomita T. Iwatsubo T. Baumeister R. Haass C. J. Biol. Chem. 1999; 274: 28669-28673Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar). We next analyzed the
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