Alzheimer Disease Aβ Production in the Absence of S-Palmitoylation-dependent Targeting of BACE1 to Lipid Rafts
2008; Elsevier BV; Volume: 284; Issue: 6 Linguagem: Inglês
10.1074/jbc.m808920200
ISSN1083-351X
AutoresKulandaivelu S. Vetrivel, Xavier Meckler, Ying Chen, Phuong D. Nguyen, Nabil G. Seidah, Robert Vassar, Philip C. Wong, Masaki Fukata, Maria Z. Kounnas, Gopal Thinakaran,
Tópico(s)Drug Transport and Resistance Mechanisms
ResumoAlzheimer disease β-amyloid (Aβ) peptides are generated via sequential proteolysis of amyloid precursor protein (APP) by BACE1 and γ-secretase. A subset of BACE1 localizes to cholesterol-rich membrane microdomains, termed lipid rafts. BACE1 processing in raft microdomains of cultured cells and neurons was characterized in previous studies by disrupting the integrity of lipid rafts by cholesterol depletion. These studies found either inhibition or elevation of Aβ production depending on the extent of cholesterol depletion, generating controversy. The intricate interplay between cholesterol levels, APP trafficking, and BACE1 processing is not clearly understood because cholesterol depletion has pleiotropic effects on Golgi morphology, vesicular trafficking, and membrane bulk fluidity. In this study, we used an alternate strategy to explore the function of BACE1 in membrane microdomains without altering the cellular cholesterol level. We demonstrate that BACE1 undergoes S-palmitoylation at four Cys residues at the junction of transmembrane and cytosolic domains, and Ala substitution at these four residues is sufficient to displace BACE1 from lipid rafts. Analysis of wild type and mutant BACE1 expressed in BACE1 null fibroblasts and neuroblastoma cells revealed that S-palmitoylation neither contributes to protein stability nor subcellular localization of BACE1. Surprisingly, non-raft localization of palmitoylation-deficient BACE1 did not have discernible influence on BACE1 processing of APP or secretion of Aβ. These results indicate that post-translational S-palmitoylation of BACE1 is not required for APP processing, and that BACE1 can efficiently cleave APP in both raft and non-raft microdomains. Alzheimer disease β-amyloid (Aβ) peptides are generated via sequential proteolysis of amyloid precursor protein (APP) by BACE1 and γ-secretase. A subset of BACE1 localizes to cholesterol-rich membrane microdomains, termed lipid rafts. BACE1 processing in raft microdomains of cultured cells and neurons was characterized in previous studies by disrupting the integrity of lipid rafts by cholesterol depletion. These studies found either inhibition or elevation of Aβ production depending on the extent of cholesterol depletion, generating controversy. The intricate interplay between cholesterol levels, APP trafficking, and BACE1 processing is not clearly understood because cholesterol depletion has pleiotropic effects on Golgi morphology, vesicular trafficking, and membrane bulk fluidity. In this study, we used an alternate strategy to explore the function of BACE1 in membrane microdomains without altering the cellular cholesterol level. We demonstrate that BACE1 undergoes S-palmitoylation at four Cys residues at the junction of transmembrane and cytosolic domains, and Ala substitution at these four residues is sufficient to displace BACE1 from lipid rafts. Analysis of wild type and mutant BACE1 expressed in BACE1 null fibroblasts and neuroblastoma cells revealed that S-palmitoylation neither contributes to protein stability nor subcellular localization of BACE1. Surprisingly, non-raft localization of palmitoylation-deficient BACE1 did not have discernible influence on BACE1 processing of APP or secretion of Aβ. These results indicate that post-translational S-palmitoylation of BACE1 is not required for APP processing, and that BACE1 can efficiently cleave APP in both raft and non-raft microdomains. Alzheimer disease-associated β-amyloid (Aβ) 3The abbreviations used are: Aβ, β-amyloid; APP, amyloid precursor protein; BACE1, β-site APP cleaving enzyme 1; CTF, C-terminal fragment; DHHC, Asp-His-His-Cys; DRM, detergent-resistant membrane; ELISA, enzyme-linked immunosorbent assay; FL, full-length; GPI, glycosylphosphatidylinositol; mAb, monoclonal antibody; MEF, mouse embryonic fibroblasts; PAT, protein acyltransferase; PLAP, placental alkaline phosphatase; PS, presenilin(s); WT, wild-type; IRES, internal ribosome entry site; TGN, trans-Golgi network; 3C/A, C478A/C482A/C485A; 4C/A, C474A/C478A/C482A/C485A. 3The abbreviations used are: Aβ, β-amyloid; APP, amyloid precursor protein; BACE1, β-site APP cleaving enzyme 1; CTF, C-terminal fragment; DHHC, Asp-His-His-Cys; DRM, detergent-resistant membrane; ELISA, enzyme-linked immunosorbent assay; FL, full-length; GPI, glycosylphosphatidylinositol; mAb, monoclonal antibody; MEF, mouse embryonic fibroblasts; PAT, protein acyltransferase; PLAP, placental alkaline phosphatase; PS, presenilin(s); WT, wild-type; IRES, internal ribosome entry site; TGN, trans-Golgi network; 3C/A, C478A/C482A/C485A; 4C/A, C474A/C478A/C482A/C485A. peptides are derived from the sequential proteolysis of β-amyloid precursor protein (APP) by β- and γ-secretases. The major β-secretase is an aspartyl protease, termed BACE1 (β-site APP-cleaving enzyme 1) (1Vassar 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: 735-741Google Scholar, 2Sinha S. Anderson J.P. Barbour R. Basi G.S. Caccavello R. Davis D. Doan M. Dovey H.F. Frigon N. Hong J. Jacobson-Croak K. Jewett N. Keim P. Knops J. Lieberburg I. Power M. Tan H. Tatsuno G. Tung J. Schenk D. Seubert P. Suomensaari S.M. Wang S. Walker D. Zhao J. McConlogue L. John V. Nature.. 1999; 402: 537-540Google Scholar, 3Yan R. Bienkowski M.J. Shuck M.E. Miao H. Tory M.C. Pauley A.M. Brashier J.R. Stratman N.C. Mathews W.R. Buhl A.E. Carter D.B. Tomasselli A.G. Parodi L.A. Heinrikson R.L. Gurney M.E. Nature.. 1999; 402: 533-537Google Scholar, 4Cai H. Wang Y. McCarthy D. Wen H. Borchelt D.R. 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Waguri S. Uchiyama Y. Wakatsuki S. Traffic.. 2004; 5: 437-448Google Scholar, 28He X. Li F. Chang W.P. Tang J. J. Biol. Chem.. 2005; 280: 11696-11703Google Scholar, 29Wahle T. Prager K. Raffler N. Haass C. Famulok M. Walter J. Mol. Cell. Neurosci.. 2005; 29: 453-461Google Scholar, 30Wahle T. Thal D.R. Sastre M. Rentmeister A. Bogdanovic N. Famulok M. Heneka M.T. Walter J. J. Neurosci.. 2006; 26: 12838-12846Google Scholar, 31Tesco G. Koh Y.H. Kang E.L. Cameron A.N. Das S. Sena-Esteves M. Hiltunen M. Yang S.H. Zhong Z. Shen Y. Simpkins J.W. Tanzi R.E. Neuron.. 2007; 54: 721-737Google Scholar). Over the years, a functional relationship between cellular cholesterol level and Aβ production has been uncovered, raising the intriguing possibility that cholesterol levels may determine the balance between amyloidogenic and non-amyloidogenic processing of APP (32Simons M. Keller P. De Strooper B. Beyreuther K. Dotti C.G. Simons K. Proc. Natl. Acad. Sci. U. S. 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Chem.. 2005; 280: 25892-25900Google Scholar). Moreover, it was recently reported that moderate reduction of cholesterol ( 35%), consistent with earlier studies (32Simons M. Keller P. De Strooper B. Beyreuther K. Dotti C.G. Simons K. Proc. Natl. Acad. Sci. U. S. A.. 1998; 95: 6460-6464Google Scholar, 33Ehehalt R. Keller P. Haass C. Thiele C. Simons K. J. Cell Biol.. 2003; 160: 113-123Google Scholar). Nevertheless, given the pleiotropic effects of cholesterol depletion on membrane properties and vesicular trafficking of secretory and endocytic proteins (42Rodal S.K. Skretting G. Garred O. Vilhardt F. van Deurs B. Sandvig K. Mol. Biol. Cell.. 1999; 10: 961-974Google Scholar, 43Hansen G.H. Niels-Christiansen L.L. Thorsen E. Immerdal L. Danielsen E.M. J. Biol. Chem.. 2000; 275: 5136-5142Google Scholar, 44Wang Y. Thiele C. Huttner W.B. Traffic.. 2000; 1: 952-962Google Scholar, 45Kirsch C. Eckert G.P. Mueller W.E. Biochem. Pharmacol.. 2003; 65: 843-856Google Scholar, 46Hao M. Mukherjee S. 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A BACE1 mutant with Ala substitution of all four Cys residues (BACE1-4C/A) fails to associate with DRM in cultured cells, but is not otherwise different from wtBACE1 in terms of protein stability, maturation, or subcellular localization. Surprisingly, APP processing and Aβ generation were unaffected in cells stably expressing the BACE1-4C/A mutant. Finally, we observed an increase in the levels of APP CTFs in detergent-soluble fractions of BACE1-4C/A as compared with wtBACE1 cells. Thus, our data collectively indicate a non-obligatory role of S-palmitoylation and lipid raft localization of BACE1 in amyloidogenic processing of APP. cDNA Constructs—Plasmids encoding C-terminal FLAG-tagged wtBACE1 and 3C/A (C478A/C482A/C485A) (19Benjannet S. Elagoz A. Wickham L. Mamarbachi M. Munzer J.S. Basak A. Lazure C. Cromlish J.A. Sisodia S. Checler F. Chretien M. Seidah N.G. J. Biol. Chem.. 2001; 276: 10879-10887Google Scholar) and hemagglutinin-tagged Asp-His-His-Cys (DHHC)-rich protein acyltransferases (PATs) have been described (48Fukata M. Fukata Y. Adesnik H. Nicoll R.A. Bredt D.S. Neuron.. 2004; 44: 987-996Google Scholar). A plasmid containing placental alkaline phosphatase (PLAP) cDNA was obtained from ATCC (clone MGC-5096). BACE1-3C/A cDNA was used as the template to generate BACE1-4C/A (C474A/C478A/C482A/C485A) by PCR mutagenesis, and the amplified segment was verified by sequencing. BACE1-GPI cDNA was constructed by overlap PCR by replacing the transmembrane and C-terminal sequences of BACE1 with the GPI anchor domain from PLAP. For retroviral expression, the cDNAs were subcloned into retroviral vector, pMXpuro (provided by Dr. Toshio Kitamura, University of Tokyo, Japan) or pLHCX (Clonetech). To construct a retroviral vector for low-level transgene expression (pMXpuroIRES), we cloned the internal ribosome entry site (IRES) from pIRES (Clontech) downstream of the puromycin resistance cassette in pMX vector. BACE1 cDNAs were then subcloned downstream of the IRES. Retroviral Infections and Generation of Stable Cell Lines—BACE1-/- mouse embryonic fibroblasts (MEF) have been previously described (49Li T. Ma G. Cai H. Price D.L. Wong P.C. J. Neurosci.. 2003; 23: 3272-3277Google Scholar). N2a cells stably expressing c-Myc epitope-tagged wtAPP (N2a 695.13) and APPSwe (N2a Swe.10) have been described previously (24Thinakaran G. Teplow D.B. Siman R. Greenberg B. Sisodia S.S. J. Biol. Chem.. 1996; 271: 9390-9397Google Scholar). The Plat-E retroviral packaging cell line was kindly provided by Dr. Toshio Kitamura (University of Tokyo, Japan). Retroviral infections were performed as described previously (50Onishi M. Kinoshita S. Morikawa Y. Shibuya A. Phillips J. Lanier L.L. Gorman D.M. Nolan G.P. Miyajima A. Kitamura T. Exp. Hematol.. 1996; 24: 324-329Google Scholar). Briefly, retroviral supernatants collected 48 h after transfection of Plat-E cells with expression vectors were used to infect BACE1-/- MEF or N2a cells in the presence of 10 μg/ml Polybrene. Stably transduced pools of MEF, N2a 695.13, or Swe.10 cells were selected in the presence of 4 μg/ml puromycin or hygromycin (400 μg/ml). Antibodies—BACE1 was detected using rabbit polyclonal antibodies anti-BACE1 (residues 46–163 (4Cai H. Wang Y. McCarthy D. Wen H. Borchelt D.R. Price D.L. Wong P.C. Nat. Neurosci.. 2001; 4: 233-234Google Scholar)) and 7523 (residues 46–60 (16Capell A. Steiner H. Willem M. Kaiser H. Meyer C. Walter J. Lammich S. Multhaup G. Haass C. J. Biol. Chem.. 2000; 275: 30849-30854Google Scholar)) (provided by Dr. Christian Haass, Ludwig-Maximilians-University, Munich), or monoclonal antibody (mAb) BACE1-Cat1 (residues 46–460 (8Zhao J. Fu Y. Yasvoina M. Shao P. Hitt B. O'Connor T. Logan S. Maus E. Citron M. Berry R. Binder L. Vassar R. J. Neurosci.. 2007; 27: 3639-3649Google Scholar)) and anti-FLAG M2 (Sigma). Rabbit polyclonal antiserum CTM1 was raised against a synthetic peptide corresponding to the C-terminal 15 amino acids of APP followed by the c-Myc epitope (MEQKLISEEDLN). Rabbit polyclonal PS1NT antiserum (residues 1–65) has been described (51Thinakaran G. Regard J.B. Bouton C.M. Harris C.L. Price D.L. Borchelt D.R. Sisodia S.S. Neurobiol. Dis.. 1998; 4: 438-453Google Scholar). Both mAb 26D6 (52Lamb B.A. Bardel K.A. Kulnane L.S. Anderson J.J. Holtz G. Wagner S.L. Sisodia S.S. Hoeger E.J. Nat. Neurosci.. 1999; 2: 695-697Google Scholar) and B436 (53Vetrivel K.S. Gong P. Bowen J.W. Cheng H. Chen Y. Carter M. Nguyen P.D. Placanica L. Wieland F.T. Li Y.M. Kounnas M.Z. Thinakaran G. Mol. Neurodegener.. 2007; 2: 4Google Scholar) (epitopes within residues 1–12 of Aβ) react with the NH2-terminal region of Aβ and also recognize sAPPα; mAb B113 is selective for Aβ40 (53Vetrivel K.S. Gong P. Bowen J.W. Cheng H. Chen Y. Carter M. Nguyen P.D. Placanica L. Wieland F.T. Li Y.M. Kounnas M.Z. Thinakaran G. Mol. Neurodegener.. 2007; 2: 4Google Scholar). The following mAbs were purchased from commercial sources: GM130, syntaxin 6, flotillin-2, γ-adaptin, EEA1 (BD Biosciences), anti-hemagglutinin (clone 16B12) and 4G8 (Covance), and mAb 5228 (Chemicon). Lipid Raft Fractionation—Lipid rafts were isolated from 0.5% Lubrol WX (Lubrol 17A17; Serva) lysates of cultured cells by discontinuous flotation density gradients as described previously (40Vetrivel K.S. Cheng H. Lin W. Sakurai T. Li T. Nukina N. Wong P.C. Xu H. Thinakaran G. J. Biol. Chem.. 2004; 279: 44945-44954Google Scholar, 41Vetrivel 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: 25892-25900Google Scholar). For the analysis of cell surface rafts, subconfluent cultures were surface biotinylated with NHS S-S biotin (Pierce) as described previously (24Thinakaran G. Teplow D.B. Siman R. Greenberg B. Sisodia S.S. J. Biol. Chem.. 1996; 271: 9390-9397Google Scholar) and then subjected to lipid raft fractionation. Cell surface-biotinylated proteins in gradient fractions were captured with streptavidin beads (Pierce) and analyzed by immunoblotting. For quantifications, optimal exposures of Western blots were analyzed by standard densitometry, and a transmission calibration step tablet (Stouffer Industries, Inc.) was used to convert raw optical densities to relative fold-differences in signal intensity using Metamorph software (Molecular Devices). Protein Analyses—Metabolic and pulse-chase labeling using [35S]Met/Cys were performed essentially as described (24Thinakaran G. Teplow D.B. Siman R. Greenberg B. Sisodia S.S. J. Biol. Chem.. 1996; 271: 9390-9397Google Scholar, 53Vetrivel K.S. Gong P. Bowen J.W. Cheng H. Chen Y. Carter M. Nguyen P.D. Placanica L. Wieland F.T. Li Y.M. Kounnas M.Z. Thinakaran G. Mol. Neurodegener.. 2007; 2: 4Google Scholar). To assess the stability of BACE1, parallel dishes were pulse-labeled for 30 min with 250 μCi/ml [35S]Met/Cys (MP Biomedicals) and chased for various time points. BACE1 was immunoprecipitated from cell lysates using anti-BACE1 antibody. For analysis of APP, cells were pulse-labeled for 15 min or continuously labeled for 3 h. Full-length APP and APP CTFs were immunoprecipitated from cell lysates using CTM1 antibody. Aβ and p3 fragments were immunoprecipitated from the conditioned medium using mAb 4G8. β-CTFs (starting at +1 residue of Aβ) were identified by probing the blots with mAb 26D6. Aβ, sAPPα, and sAPPβ Measurements—Conditioned media were collected 48 h after plating the cells and the levels of secreted Aβ and sAPPα were quantified by ELISA as described previously (53Vetrivel K.S. Gong P. Bowen J.W. Cheng H. Chen Y. Carter M. Nguyen P.D. Placanica L. Wieland F.T. Li Y.M. Kounnas M.Z. Thinakaran G. Mol. Neurodegener.. 2007; 2: 4Google Scholar). Aβ1–40, Aβx-40, and Aβ1-x were measured using specific sandwich ELISAs. Aβ peptides were captured using mAb B113 for Aβ1–40 and Aβx-40 ELISA, and mAb B436 for Aβ1-x ELISA. Bound peptides were detected using alkaline phosphatase-conjugated mAb B436 for Aβ1–40 ELISA or biotinylated mAb 4G8 in combination with the streptavidin-alkaline phosphatase complex for Aβx-40 and Aβ1-x ELISA. Alkaline phosphatase activity was measured using CSPD-Sapphire II Luminescence Substrate (Applied Biosystems) and relative luminescence unit values were measured using a standard 96-well luminometer. Each sample was assayed in duplicate using appropriate dilution of the conditioned media so that the relative luminescent units were in the linear range of the standards included on each plate. Synthetic Aβ40 peptide (Bachem) was diluted in culture medium to generate standard curve (ranging from 1 to 1000 pg/well). sAPPα was quantified by sandwich ELISA using mAb 5228 for capture and mAb B436 for detection, and quantified using a standard curve prepared using affinity-purified sAPPα as described (53Vetrivel K.S. Gong P. Bowen J.W. Cheng H. Chen Y. Carter M. Nguyen P.D. Placanica L. Wieland F.T. Li Y.M. Kounnas M.Z. Thinakaran G. Mol. Neurodegener.. 2007; 2: 4Google Scholar). sAPPβ was quantified using a commercial sβAPP wild-type ELISA kit and Meso Scale Sector Imager 6000 (Meso Scale Discovery, Gaithersburg, MD) for detection following the manufacturer's recommended protocol. Captured sAPPβ was quantified by comparing the signals of the samples to a standard curve included on each plate prepared using recombinant sAPPβ in complete medium. Analysis of BACE1 Palmitoylation—COS7 cells were cotransfected with BACE1 and DHHC plasmids using Lipofectamine 2000 (Invitrogen) and labeled 48 h after transfection. Stable pools of BACE1-/- MEF and N2a cells were grown to subconfluence prior to labeling. Cells were preincubated for 1 h in Dulbecco's modified Eagle's medium supplemented with 1 mg/ml fatty acid-free bovine serum albumin (Sigma) and labeled for 4 h with 0.5 mCi/ml [10,11-3H]palmitic acid (American Radiolabeled Chemicals) diluted in the preincubation medium. Cells were scrapped in lysis buffer (150 mm NaCl, 50 mm Tris-HCl, pH 7.4, 0.5% Nonidet P-40, 0.5% sodium deoxycholate, 5 mm EDTA, 0.25% SDS, 0.25 mm phenylmethylsulfonyl fluoride, Roche Protease Inhibitor Mixture 1X), and sonicated for 30 s on ice. Aliquots of lysates (adjusted to trichloroacetic acid precipitable radioactivity) were incubated overnight with 2 μl of anti-FLAG M2 antibody to immunoprecipitate BACE1. Immunoprecipitates were fractionated by SDS-PAGE, transferred to polyvinylidene difluoride membrane (Bio-Rad), and [3H]palmitic acid-labeled BACE1 was detected by PhosphorImager analysis (GE Healthcare). The membranes were subsequently subject to Western blotting with FLAG M2 antibody (1:20,000) to reveal total immunoprecipitated BACE1. To compare the relative efficiencies of BACE1 palmitoylation by each DHHC, the ratio between [3H]palmitic acid-labeled BACE1 and immunoblot BACE1 signal intensities were quantified using Image J software. Immunofluorescence Microscopy—Cells cultured on poly-l-lysine-coated coverslips were fixed using 4% paraformaldehyde. Polyclonal BACE1 antiserum 7523 and mAb against γ-adaptin or transferrin receptor were diluted in phosphate-buffered saline containing 3% bovine serum albumin and 0.2% Tween 20, and incubated with fixed cells at room temperature for 2 h. Images were acquired on a Zeiss confocal microscope (Pascal) using ×100 1.45 NA Plan-Apochromat oil objective. Images were processed using Metamorph software (Molecular Devices). BACE1 Is S-Palmitoylated at 4 Cysteine Residues—Previously it was reported that BACE
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