Functional Overexpression of γ-Secretase Reveals Protease-independent Trafficking Functions and a Critical Role of Lipids for Protease Activity
2004; Elsevier BV; Volume: 280; Issue: 13 Linguagem: Inglês
10.1074/jbc.m413086200
ISSN1083-351X
AutoresJonathan D.J. Wrigley, Irina Schurov, Emma J. Nunn, Agnés C. L. Martin, Earl E. Clarke, Semantha Ellis, Timothy P. Bonnert, Mark S. Shearman, Dirk Beher,
Tópico(s)Lipid Membrane Structure and Behavior
ResumoPresenilins appear to form the active center of γ-secretase but require the presence of the integral membrane proteins nicastrin, anterior pharynx defective 1, and presenilin enhancer 2 for catalytic function. We have simultaneously overexpressed all of these polypeptides, and we demonstrate functional assembly of the enzyme complex, a substantial increase in enzyme activity, and binding of all components to a transition state analogue γ-secretase inhibitor. Co-localization of all components can be observed in the Golgi compartment, and further trafficking of the individual constituents seems to be dependent on functional assembly. Apart from its catalytic function, γ-secretase appears to play a role in the trafficking of the β-amyloid precursor protein, which was changed upon reconstitution of the enzyme but unaffected by pharmacological inhibition. Because the relative molecular mass and stoichiometry of the active enzyme complex remain elusive, we performed size exclusion chromatography of solubilized γ-secretase, which yielded evidence of a tetrameric form of the complex, yet almost completely abolished enzyme activity. γ-Secretase activity was reconstituted upon addition of an independent size exclusion chromatography fraction of lower molecular mass and nonproteinaceous nature, which could be replaced by a brain lipid extract. The same treatment was able to restore enzyme activity after immunoaffinity purification of the γ-secretase complex, demonstrating that lipids play a key role in preserving the catalytic activity of this protease. Furthermore, these data show that it is important to discriminate between intact, inactive γ-secretase complexes and the active form of the enzyme, indicating the care that must be taken in the study of γ-secretase. Presenilins appear to form the active center of γ-secretase but require the presence of the integral membrane proteins nicastrin, anterior pharynx defective 1, and presenilin enhancer 2 for catalytic function. We have simultaneously overexpressed all of these polypeptides, and we demonstrate functional assembly of the enzyme complex, a substantial increase in enzyme activity, and binding of all components to a transition state analogue γ-secretase inhibitor. Co-localization of all components can be observed in the Golgi compartment, and further trafficking of the individual constituents seems to be dependent on functional assembly. Apart from its catalytic function, γ-secretase appears to play a role in the trafficking of the β-amyloid precursor protein, which was changed upon reconstitution of the enzyme but unaffected by pharmacological inhibition. Because the relative molecular mass and stoichiometry of the active enzyme complex remain elusive, we performed size exclusion chromatography of solubilized γ-secretase, which yielded evidence of a tetrameric form of the complex, yet almost completely abolished enzyme activity. γ-Secretase activity was reconstituted upon addition of an independent size exclusion chromatography fraction of lower molecular mass and nonproteinaceous nature, which could be replaced by a brain lipid extract. The same treatment was able to restore enzyme activity after immunoaffinity purification of the γ-secretase complex, demonstrating that lipids play a key role in preserving the catalytic activity of this protease. Furthermore, these data show that it is important to discriminate between intact, inactive γ-secretase complexes and the active form of the enzyme, indicating the care that must be taken in the study of γ-secretase. γ-Secretase has been characterized as an unconventional aspartyl protease that processes type I membrane proteins after removal of their ectodomains by cleavage within their transmembrane domains. This enzyme has created great interest because it performs the final proteolytic cleavage step in the processing cascade of the β-amyloid precursor protein (βAPP). 1The abbreviations used are: βAPP, β-amyloid precursor protein; AD, Alzheimer's disease; Aβ, amyloid-β peptide; CHAPSO, 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate; CTF, C-terminal fragment; NTF, N-terminal fragment; MCD, methyl-β-cyclodextrin; MES, 2-(N-morpholino)ethanesulfonic acid; PS, presenilin; SEC, size exclusion chromatography; ER, endoplasmic reticulum. γ-Secretase cleavage leads to the production of amyloid-β (Aβ) peptides, which are generally believed to be the causative agents for Alzheimer's disease (AD). Because of its contribution to Aβ peptide production in the first instance and therefore to toxic events subsequently initiated by Aβ, inhibition of γ-secretase is also seen as a logical approach for the development of a disease-modifying treatment for this neurodegenerative condition. Extracellular clipping of βAPP by the aspartyl protease β-secretase (BACE1) (1.Vassar 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 (3327) Google Scholar, 2.Hussain I. Powell D. Howlett D.R. Tew D.G. Meek T.D. Chapman C. Gloger I.S. Murphy K.E. Southan C.D. Ryan D.M. Smith T.S. Simmons D.L. Walsh F.S. Dingwall C. Christie G. Mol. Cell Neurosci. 1999; 14: 419-427Crossref PubMed Scopus (1002) Google Scholar, 3.Sinha 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. John V. 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The requirement for the assembly of a complex consisting of at least four membrane proteins demonstrates the intricacy of the enzyme. It is not implausible, however, that other co-factors may be constituents of γ-secretase and modulate its catalytic function. The exact stoichiometry of the active complex is also elusive because not only have PS complexes of various molecular weights been described (24.Takasugi N. Tomita T. Hayashi I. Tsuruoka M. Niimura M. Takahashi Y. Thinakaran G. Iwatsubo T. Nature. 2003; 422: 438-441Crossref PubMed Scopus (789) Google Scholar, 25.Kimberly 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 (684) Google Scholar, 26.Capell A. Grunberg J. Pesold B. Diehlmann A. Citron M. Nixon R. Beyreuther K. Selkoe D.J. Haass C. J. Biol. Chem. 1998; 273: 3205-3211Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar, 27.Yu G. Chen F. Levesque G. Nishimura M. 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More recent evidence suggests a PS dimer at the core of the enzyme complex, with substrates being cleaved at the interface between the two PS molecules (31.Schroeter E.H. Ilagan M.X. Brunkan A.L. Hecimovic S. Li Y.M. Xu M. Lewis H.D. Saxena M.T. De Strooper B. Coonrod A. Tomita T. Iwatsubo T. Moore C.L. Goate A. Wolfe M.S. Shearman M. Kopan R. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 13075-13080Crossref PubMed Scopus (181) Google Scholar). During the formation of the enzyme complex, its known components must associate and undergo a maturation process to yield the functionally active complex. Several groups have detected the formation of subcomplexes of Aph-1 and nicastrin during complex formation (32.LaVoie M.J. Fraering P.C. Ostaszewski B.L. Ye W. Kimberly W.T. Wolfe M.S. Selkoe D.J. J. Biol. Chem. 2003; 278: 37213-37222Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 33.Hu Y. Fortini M.E. J. 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Apart from these findings, down-regulation or genetic inactivation of one of the components of this complex directly affects the maturation and/or stability of the other interacting factors (22.Francis R. McGrath G. Zhang J. Ruddy D.A. Sym M. Apfeld J. Nicoll M. Maxwell M. Hai B. Ellis M.C. Parks A.L. Xu W. Li J. Gurney M. Myers R.L. Himes C.S. Hiebsch R. Ruble C. Nye J.S. Curtis D. Dev. Cell. 2002; 3: 85-97Abstract Full Text Full Text PDF PubMed Scopus (714) Google Scholar, 35.Edbauer D. Winkler E. Haass C. Steiner H. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 8666-8671Crossref PubMed Scopus (218) Google Scholar, 36.Steiner H. Winkler E. Edbauer D. Prokop S. Basset G. Yamasaki A. Kostka M. Haass C. J. Biol. Chem. 2002; 277: 39062-39065Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar, 37.Luo W.J. Wang H. Li H. Kim B.S. Shah S. Lee H.J. Thinakaran G. Kim T.W. Yu G. Xu H. J. Biol. Chem. 2003; 278: 7850-7854Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar). It therefore appears that the association and maturation of these cofactors, along with the activation of the mature complex, possibly through PS endoproteolysis is an ordered and highly regulated process. To study this enzyme further, we have generated a novel cell line referred to as γ(NRC-F8)-secretase cell line, by sequentially co-expressing the substrate precursor βAPP695 and the complex components PS1, nicastrin, Aph-1aL, and Pen-2. This cell line displays the characteristics of a γ-secretase overexpressing in vitro system, manifested by changes in nicastrin maturation and PS1 endoproteolytic processing, an ∼14-fold increase in both cell-free γ(40)- and γ(42)-enzyme activities, and isolation of all components with the active site-directed (38.Wrigley J.D. Nunn E.J. Nyabi O. Clarke E.E. Hunt P. Nadin A. De Strooper B. Shearman M.S. Beher D. J. Neurochem. 2004; 90: 1312-1320Crossref PubMed Scopus (23) Google Scholar) biotinylated transition state inhibitor Merck C (39.Beher D. Fricker M. Nadin A. Clarke E.E. Wrigley J.D. Li Y.M. Culvenor J.G. Masters C.L. Harrison T. Shearman M.S. Biochemistry. 2003; 42: 8133-8142Crossref PubMed Scopus (80) Google Scholar). By having demonstrated a successful and functional overexpression of the enzyme, we utilized a direct γ-secretase substrate to investigate the potential cause of the discrepancies that have been observed with respect to cell-free and cellular Aβ peptide production in similar studies (24.Takasugi N. Tomita T. Hayashi I. Tsuruoka M. Niimura M. Takahashi Y. Thinakaran G. Iwatsubo T. Nature. 2003; 422: 438-441Crossref PubMed Scopus (789) Google Scholar, 25.Kimberly 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 (684) Google Scholar, 40.Kim S.H. Ikeuchi T. Yu C. Sisodia S.S. J. Biol. Chem. 2003; 278: 33992-34002Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Confocal immunofluorescence microscopy provided insights into the regulation of enzyme trafficking to the cell surface and implied a protease-independent role of the functional γ-secretase complex in the regulation of the maturation and trafficking of βAPP. Size exclusion chromatography (SEC) and immunoaffinity isolation have been employed to provide evidence for a tetrameric enzyme species, highlight several key considerations in the purification of γ-secretase, and most significantly to demonstrate that lipids are critical for preservation of the active conformation of the enzyme complex. Antibodies—Monoclonal and polyclonal antibodies were obtained from the following sources and diluted for Western blot analyses as indicated: anti-β-catenin (monoclonal mouse, BD Biosciences, 1:1000), anti-FLAG (monoclonal mouse, Sigma, 1:5000), anti-V5 (monoclonal mouse, Invitrogen, 1:1000), anti-nicastrin (rabbit polyclonal, Sigma, 1:10,000), anti-α-tubulin (monoclonal mouse, Sigma, 1:5000), and horseradish peroxidase-conjugated polyclonal goat anti-mouse and anti-rabbit F(ab′)2 fragments (Amersham Biosciences, 1:5000). PS1-FL and its fragments were detected by using rabbit polyclonal antiserum 00/2 raised against the loop peptide 301–317 (41.Evin G. Sharples R.A. Weidemann A. Reinhard F.B. Carbone V. Culvenor J.G. Holsinger R.M. Sernee M.F. Beyreuther K. Masters C.L. Biochemistry. 2001; 40: 8359-8368Crossref PubMed Scopus (43) Google Scholar) (1:2000) and polyclonal antiserum 98/1 raised against residues 1–20 of PS1 (1:2500) (42.Culvenor J.G. Evin G. Cooney M.A. Wardan H. Sharples R.A. Maher F. Reed G. Diehlmann A. Weidemann A. Beyreuther K. Masters C.L. Exp. Cell Res. 2000; 255: 192-206Crossref PubMed Scopus (38) Google Scholar). βAPP was detected by using polyclonal rabbit antiserum R7334 raised against residues 659–694 of βAPP695 (28.Beher D. Wrigley J.D. Nadin A. Evin G. Masters C.L. Harrison T. Castro J.L. Shearman M.S. J. Biol. Chem. 2001; 276: 45394-45402Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Antibodies for immunocytochemistry were diluted as follows: anti-FLAG (Sigma, 1:2000); anti-V5 (Invitrogen, 1:200); anti-nicastrin (Sigma, 1:1000); anti-Golgi 58K protein clone 58K-9 (Sigma, 1:500); anti-Pan-cadherin (CH-19, Abcam, 1:200); and anti-PS1 (98/1, 1:500), fluorescein isothiocyanate-, Cy3-, and Cy5-conjugated secondary antibodies (Jackson ImmunoResearch, 1:100). βAPP was detected using antibodies either directed against the ectodomain, monoclonal antibody 22C11 (residues 61–81, Chemicon, 1:1000) and a polyclonal antibody against residues 1–100 (mAbP2–1, Affinity Bioreagents, 1:500), or polyclonal antibodies against the C terminus, residues 751–770 of βAPP770 (Calbiochem, 1:1000) and residues around Y668 βAPP695 (Cell Signaling, 1:500). Complementary DNA Constructs—Expression vectors encoding human nicastrin and Aph-1aL with a C-terminal V5-hexahistidine epitope tag were generated by assembling IMAGE clones (43.Lennon G. Auffray C. Polymeropoulos M. Soares M.B. Genomics. 1996; 33: 151-152Crossref PubMed Scopus (1089) Google Scholar) encoding the human sequences. Subcloning in the respective pcDNA vectors (Invitrogen) yielded the nicastrin-pcDNA3.1/Hygro(+) and Aph-1aL-pcDNA-3.1/V5-His/Neo(+) constructs that were confirmed by DNA sequencing. The generation of Pen-2-pcDNA3.1/FLAG/Neo(+) vector has been described (44.Bergman A. Hansson E. Pursglove S.E. Farmery M.R. Lannfelt L. Lendahl U. Lundkvist J. Naslund J. J. Biol. Chem. 2004; 279: 16744-16753Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Generation of Stable Cell Lines—HEK cell lines stably overexpressing βAPP695 alone or in combination with human PS1 have been described previously (38.Wrigley J.D. Nunn E.J. Nyabi O. Clarke E.E. Hunt P. Nadin A. De Strooper B. Shearman M.S. Beher D. J. Neurochem. 2004; 90: 1312-1320Crossref PubMed Scopus (23) Google Scholar). For sequential introduction of additional γ-secretase complex components, the βAPP695/PS1 cell line (clone B9) was transfected with nicastrin-pcDNA3.1/Hygro(+) using standard calcium phosphate methods. Transfectants were cultured in the presence of 1 μg/ml puromycin (Sigma) and 100 μg/ml Zeocin (Invitrogen) to maintain βAPP and PS1 expression, respectively, and 500 μg/ml hygromycin (Invitrogen) to select for nicastrin expression (as determined from a kill curve carried out with the founder βAPP/PS1 cells). After dilution, individual surviving colonies were picked and screened for the expression of βAPP695, PS1, and nicastrin by Western blotting. The strongest nicastrin-overexpressing cell line (clone D8) was expanded for the next round of transfections. For this instance, Aph-1aL-pcDNA3.1/V5-His/Neo(+) and Pen-2-pcDNA3.1/FLAG/Neo(+) were transfected simultaneously into clone D8 using the Gene Juice transfection reagent (Novagen) according to the manufacturer's instructions. Transfectants were selected for expression of Aph-1aL and Pen-2 with 5 mg/ml of geneticin (Invitrogen) (as determined from a kill curve carried out with the founder βAPP/PS1/nicastrin cell line). After dilution, individual surviving colonies were picked and screened for the expression of all polypeptides by Western blotting. Clone F8, referred to as the γ(NRC-F8)-secretase cell line, was chosen for expansion because it showed the strongest expression of all exogenous polypeptides. Transient Transfections and Solubilization of βAPP-CTFs—Cells were plated at 2 × 106 cells/10-cm dish and transfected on the following day with 5 μg of SPA4CT cDNA (45.Dyrks T. Dyrks E. Monning U. Urmoneit B. Turner J. Beyreuther K. FEBS Lett. 1993; 335: 89-93Crossref PubMed Scopus (96) Google Scholar) or empty vector (mock control) using Gene Juice transfection reagent according to the manufacturer's instructions (Novagen). The cells were harvested after 2 days and triturated in 1 ml of TBS (50 mm Tris-HCl, pH 7.4, 150 mm NaCl). Membranes were collected by centrifugation for 30 min at 65,000 rpm in a TLA-100.2 rotor (Beckman) at 4 °C. Membrane proteins were solubilized in TBS, 1% Triton X-100, 1× EDTA-free protease inhibitor mixture (Roche Applied Science) by trituration. After incubation for 30 min on ice, insoluble debris was removed by centrifugation, and the samples were processed for Western blotting as described below. Extraction of Protein from Whole Cells for Western Blot Analysis— Cells were collected in phosphate-buffered saline (PBS) followed by centrifugation for 10 min at 6000 × g at 4 °C. Cell lysis was performed by incubation for 25 min under constant shaking in 50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1% Triton X-100, 0.5% Nonidet P-40, 0.2% SDS, 1 mm EDTA, 1× EDTA-free protease inhibitor mixture (Roche Applied Science) at 4 °C. Insoluble debris was removed by centrifugation for 10 min at 20,000 × g, and protein levels were determined by using the bicinchoninic acid assay in a 96-well plate format according to the manufacturer's instructions (Pierce and Warriner). Equal amounts of protein were separated by SDS-PAGE and transferred to nitrocellulose membranes. Probing of the membranes was carried out as described previously (28.Beher D. Wrigley J.D. Nadin A. Evin G. Masters C.L. Harrison T. Castro J.L. Shearman M.S. J. Biol. Chem. 2001; 276: 45394-45402Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar) with various antibodies, as indicated in the figure legends, using the enhanced-chemiluminescence system (ECL, Amersham Biosciences). Immunocytochemistry—85 × 103 cells/ml were seeded on polylysine-coated glass coverslips the day before the experiment and washed with PBS, fixed with 4% paraformaldehyde, and permeabilized for 10 min at 4 °C with 0.5% Triton X-100. After blocking for 1 h at room temperature with 10% normal goat serum (Sigma), the cells were incubated for 1 h at room temperature with primary antiserum followed by a fluorophore-conjugated secondary antibody (Jackson ImmunoResearch). For co-immunostaining, the cells were further incubated with a second primary and secondary antibody conjugated to an alternative fluorophore. The cells were washed with PBS and nuclei stained with TOTO-3 iodine for 30 s (Molecular Probes). The coverslips were mounted onto glass slides using Vectashield mounting medium (Vector Laboratories), and images were collected on a Leica confocal microscope using a Leica TCS NT "Image" program version 1.6.587. Quantification of Aβ Peptides in Conditioned Cell Culture Media— Cells were plated at 6 × 106 cells/10-cm dish, and the media were exchanged on the following day. Aβ peptide secretion into the fresh media after overnight incubation was quantified by an Origen™ electrochemiluminescence assay (Origen M-Series™ analyzer, Igen) as described previously (28.Beher D. Wrigley J.D. Nadin A. Evin G. Masters C.L. Harrison T. Castro J.L. Shearman M.S. J. Biol. Chem. 2001; 276: 45394-45402Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Membrane Preparation for Affinity Precipitations and Cell-free γ-Secretase Assays—Membranes from the stable cell lines were prepared essentially as described previously (28.Beher D. Wrigley J.D. Nadin A. Evin G. Masters C.L. Harrison T. Castro J.L. Shearman M.S. J. Biol. Chem. 2001; 276: 45394-45402Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Briefly, after collection in phosphate-buffered saline (PBS), 2 mm EDTA cells were hypotonically shocked by incubation for 8 min in 20 mm HEPES-HCl, pH 7.3, 10 mm KCl and sedimented by centrifugation for 10 min at 1000 × g. Cells were homogenized in 20 mm HEPES-HCl, pH 7.3, 90 mm KCl, and nuclei and cellular debris were removed by centrifugation for 10 min at 1000 × g. Cellular membranes were collected by centrifugation for 1 h at 45,000 rpm (50.2 Ti rotor, Beckman), resuspended in PBS, 5% glycerol, and stored at –80 °C prior to further use. CHAPSO Solubilization of Active γ-Secretase—Cell membranes (stored in PBS, 5% glycerol) were collected by centrifugation for 30 min at 180,000 × g. Membrane proteins were solubilized in 1% (w/v) CHAPSO, 50 mm MES-NaOH, pH 6.0, 1 mm EDTA, 0.15 m NaCl, 5 mm MgCl2, 1× EDTA-free protease inhibitor mixture (Roche Applied Science). Insoluble debris was removed by centrifugation at
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