In vitro gamma-secretase cleavage of the Alzheimer’s amyloid precursor protein correlates to a subset of presenilin complexes and is inhibited by zinc David E Hoke, Jiang-Li Tan, Nancy T Ilaya, Janetta G Culvenor, Stephanie J Smith, ` Anthony R White, Colin L Masters and Genevieve M Evin Department of Pathology, The University of Melbourne and the Mental Health Research Institute, Parkville, Victoria, Australia Keywords Alzheimer’s disease; amyloid precursor protein; gamma-secretase; amyloid beta Correspondence D E Hoke, Department of Microbiology, Monash University, Clayton, Vic 3800, Australia Fax: +61 39905 4811 Tel: +61 39905 4807 E-mail: david.hoke@med.monash.edu.au G M Evin, Department of Pathology, The University of Melbourne, Parkville, Vic 3010, Australia Fax: +61 38344 4004 Tel: +61 38344 4205 E-mail: gmevin@unimelb.edu.au (Received 20 May 2005, revised August 2005, accepted 30 August 2005) doi:10.1111/j.1742-4658.2005.04950.x The c-secretase complex mediates the final proteolytic event in Alzheimer’s disease amyloid-b biogenesis This membrane complex of presenilin, anterior pharynx defective, nicastrin, and presenilin enhancer-2 cleaves the C-terminal 99-amino acid fragment of the amyloid precursor protein intramembranously at c-sites to form C-terminally heterogeneous amyloid-b and cleaves at an e-site to release the intracellular domain or e-C-terminal fragment In this work, two novel in vitro c-secretase assays are developed to further explore the biochemical characteristics of c-secretase activity During development of a bacterial expression system for a substrate based on the amyloid precursor protein C-terminal 99-amino acid sequence, fragments similar to amyloid-b and an e-C-terminal fragment were observed Upon purification this substrate was used in parallel with a transfected source of substrate to measure c-secretase activity from detergent extracted membranes With these systems, it was determined that recovery of sizefractionated cellular and tissue-derived c-secretase activity is dependent upon detergent concentration and that activity correlates to a subset of high molecular mass presenilin complexes We also show that by changing the solvent environment with dimethyl sulfoxide, detection of e-C-terminal fragments can be elevated Lastly, we show that zinc causes an increase in the apparent molecular mass of an amyloid precursor protein c-secretase substrate and inhibits its cleavage These studies further refine our knowledge of the complexes and biochemical factors needed for c-secretase activity and suggest a mechanism by which zinc dysregulation may contribute to Alzheimer’s disease pathogenesis Gamma-secretase is an aspartyl protease that cleaves type I integral membrane proteins intramembranously The amyloid precursor protein (APP) undergoes sequential cleavages by beta-site APP cleaving enzyme and c-secretase to form amyloid-b (Ab) The beta-site APP cleaving enzyme cleavage releases an APP ectodomain leaving a 99-amino acid membrane spanning C-terminal fragment (CTF), C99 C99 then undergoes intramembranous cleavage to form Ab peptides of different lengths c-secretase also releases an APP intracellular domain (AICD or e-CTF) by cleaving 9–7 amino acids from c 40 and 42 sites at the e site [1–3] Several lines of evidence support the pathogenic role of c-cleavage of APP in Alzheimer’s disease (AD) The genes encoding presenilin and (PS) are essential for c-secretase activity and 150 mutations in the PS genes have been found associated with autosomal dominant early onset familial AD [4] Although only 16 mutations have been found in the APP gene, the majority linked to early onset familial AD occur in the Abbreviations AD, Alzheimer’s disease; APP, amyloid precursor protein; CTF, C-terminal fragment; NTF, N-terminal fragment; PS, presenilin 5544 FEBS Journal 272 (2005) 5544–5557 ª 2005 FEBS D E Hoke et al transmembrane region near c and e cleavage sites (as reviewed in [5]) Gamma-secretase activity is attributed to an integral membrane complex of the four transmembrane proteins: PS, nicastrin (Nct), anterior pharynx-defective, and presenilin enhancer (as reviewed in [6]) In vitro c-secretase assays have been essential in elucidating the mechanism of inhibitors [7–9], the structure of active c-secretase complexes [10,11], and have aided in the finding of activity-modulating factors [12,13] These assays have shown that peripheral membrane proteins are not necessary for activity as carbonate washing retains activity [14] Additionally, detergent solubilization has allowed solution-based biochemical manipulation to show that all four of the genetically determined c-secretase components interact to form high molecular mass, enzymatically active c-secretase complexes [10,11,15] In this paper we describe two novel in vitro c-secretase assays that differ in substrate and enzyme source to monitor c-secretase activity without the need to overexpress the c-secretase complex components These assays are used to test the effects of detergent concentration, solvents and metals on the c-secretase cleavage of APP substrates These studies show that extracts from Escherichia coli transformed with a c-secretase substrate contain products similar to those expected from c-secretase cleavage Furthering the characterization of c-secretase activity, we show that the detergent concentration used during gel filtration affects the recovery of activity These studies also show that dimethylsulfoxide is a solvent that allows greater detection of c-secretase activity Lastly, zinc causes structural changes in a c-secretase substrate and acts as an inhibitor of csecretase cleavage of APP Results Design of a novel APP c-secretase substrate and standards Sensitive western blot assays for c-secretase were based on the production of a 3FLAG-tagged e-CTF from an APP substrate An E coli expression vector was made to encode a starting methionine, the C-terminal 99 amino acids of APP and a C-terminal triple FLAG tag The resulting protein was named MC993FLAG (Fig 1A) Escherichia coli expression vectors encoding 3FLAG-tagged APP-CTFs mimicking products from cleavage at position 40 (gamma-3FLAG standard) and 49 (epsilon-3FLAG standard) were also made to aid in the identification of 3FLAG-tagged CTFs (Fig 1A) FEBS Journal 272 (2005) 5544–5557 ª 2005 FEBS Characterization of in vitro c-secretase activity Ab-like and e-CTF-like products are present in extracts from E coli expressing MC99-3FLAG Upon expression of MC99-3FLAG in E coli, we observed three predominant anti-FLAG immunoreactive peptides The major product migrated at  18 kDa and was also detected by anti-Ab antibody, WO2 From its apparent molecular mass and its immunoreactivity, it can be concluded that it corresponds to MC99-3FLAG (Fig 1B and C) There were also several higher molecular mass and degraded species identified by both antibodies The higher molecular mass forms may correspond to aggregated MC99-3FLAG One anti-FLAG immunoreactive peptide migrated similarly to the gamma and epsilon standards at 9 kDa (Fig 1B) and the corresponding N-terminal fragment resembling Ab was identified by WO2 western blot analysis (Fig 1C) Further identification of the anti-FLAG-immunoreactive CTFs was made by coelectrophoresis with gamma and epsilon standards Co-electrophoresis obviates subtle lane-to-lane variations that may occur for these low molecular mass proteins The  9-kDa peptide comigrated with the e-3FLAG standard (Fig 1D) but faster than the c-3FLAG standard (Fig 1E) No anti-FLAG immunoreactivity was detected in lysates of mock-transformed cells (Fig 1E) Collectively, these data indicate that upon expression or during purification, a small fraction of MC99-3FLAG is degraded into multiple species including peptides resembling products expected from c-secretase cleavage Development of in vitro c-secretase assays using purified MC99-3FLAG as a substrate Peptides similar to an e-CTF present in substrate preparations would interfere with the detection of e-CTF production from mammalian tissue extracts Therefore a purification strategy was devised to minimize this contamination An initial nondenaturing size-exclusion chromatography step was performed to separate MC99-3FLAG from lower molecular mass fragments Unexpectedly, MC99-3FLAG eluted at the void volume of the column while fragments eluted according to their apparent molecular mass determined by SDS ⁄ PAGE and western blot analysis (data not shown) These MC99-3FLAG enriched void volume fractions were purified in a second-step by anti-FLAG chromatography This two-step purified material was used in c-secretase assays MC99-3FLAG was tested for cleavage by c-secretase from PS1A246E transgenic mouse brain [16] (Fig 2A) prepared by solubilization of carbonate-washed membranes with 5545 Characterization of in vitro c-secretase activity D E Hoke et al A B D C E 1% [3-[(3-cholamidopropyl)dimethylammonio-]-2-hydroxy-1-propanesulfonate] (CHAPSO) Upon incubation at 37 °C, generation of an 9 kDa CTF was detected by western blotting with anti-FLAG antibody The c-secretase inhibitor L-685,458 was used to confirm that the fragment detected was produced by c-secretase activity L-685,458 inhibited formation of this e-CTF-3FLAG in a dose-dependent manner, with an effect still observed at concentrations as low as 3.3 nm, consistent with previous reports [17] The preparation of MC99-3FLAG contained additional anti-FLAG immunoreactive peptides but these did not interfere with the assay (Fig 2A, long exposure) 5546 Fig Escherichia coli produces peptides similar to Ab and an e-CTF when transformed with MC99-3FLAG (A) Schematic of proteins (B) Lysate from MC99-3FLAGexpressing E coli, c and e standards were separated by SDS ⁄ PAGE and western blotted for FLAG immunoreactivity MC993FLAG migrates between the 20 and 14-kDa molecular mass markers One lower molecular mass FLAG immunoreactive protein has a mobility similar to the c and e standards (C) Lysate from E coli transformed with the MC99-3FLAG expression vector was probed with monoclonal antibody WO2, directed to the N-terminal region of Ab Besides MC99-3FLAG, this antibody detected several peptides of higher and lower molecular masss, one of them with a similar mobility as synthetic Ab40 (D) AntiFLAG western blot analysis of lysate from MC99-3FLAG-transformed E coli alone or spiked with different amounts of the e-3FLAG standard An uncharacterized antiFLAG immunoreactive protein migrating just below the e-CTF-like peptide is indicated by * Note that ‘e std.’ migrated identically to the E coli product marked ‘e’ (E) A similar experiment to that shown in (D) was performed by spiking MC99-3FLAG-transformed E coli lysate with c-3FLAG standard This standard migrated slower than the E coli product Mock-transformed E coli had no background anti-FLAG immunoreactivity Assay sensitivity was tested by varying the enzyme amount, enzyme dilution, and CHAPSO concentration For this experiment, c-secretase activity was prepared by extracting carbonate-washed guinea pig brain membranes with 1% CHAPSO A mgỈmL)1 extract was diluted to obtain a final concentration of 0.5% CHAPSO, and dilutions were made in 0.5% CHAPSO to 20 lgỈmL)1 Incubation of these dilutions with substrate showed c-secretase activity to be enzyme dosedependent, and that signal was detectable using the 40 lgỈmL)1 dilution of extract Therefore, as little as lg of membrane extract was sufficient to obtain a signal (Fig 2B) Secondly, the mgỈmL)1 extract was FEBS Journal 272 (2005) 5544–5557 ª 2005 FEBS D E Hoke et al A Characterization of in vitro c-secretase activity B C Fig Detection of c-secretase activity in tissue extracts using exogenous MC99-3FLAG substrate (A) Purified MC99-3FLAG substrate was added to 0.5% CHAPSO soluble c-secretase from PS1 A246E transgenic mouse brain and incubated at 37 or °C for 15 h with or without L-685,458 inhibitor These reactions were analysed by anti-FLAG western blot analysis Gamma-secretase activity was defined as the generation of e-CTF-3FLAG (e) signal upon incubation at 37 °C over background °C levels; this was inhibited by L685,458 in a dose-dependent manner Longer exposures showed a contaminating CTF (indicated by *) that migrated slightly faster than the e-CTF (B) Sensitivity of exogenous substrate c-secretase assay in the presence of 0.5% CHAPSO Guinea pig brain soluble c-secretase was diluted to 0.5% CHAPSO and further dilutions in 0.5% CHAPSO were incubated with MC99-3FLAG at 37 or °C for 15 h The reactions were analysed by anti-FLAG Western blot Gamma-secretase activity was detected using as little as lg of membrane extract (C) A similar experiment to that in (B) except that guinea pig brain soluble c-secretase was diluted to 0.25% CHAPSO with further dilutions in 0.25% CHAPSO incubated with MC99-3FLAG The most highly concentrated reaction shows little activity while the lowest concentration shows the greatest activity diluted to 0.25% CHAPSO and subsequently diluted in 0.25% CHAPSO to 40 lgỈmL)1 In contrast to the 0.5% CHAPSO dilution, 0.25% dilution did not show a dose-dependent relation of enzyme amount to product formed (Fig 2C) Rather the highest concentration and amount showed little activity while the least concentrated sample (1.2 lg of 40 lgỈmL)1) showed the highest activity and greater than the corresponding dilution in 0.5% CHAPSO Perhaps this 0.25% concentration was not enough to keep high concentrations of extract solubilized leading to an apparent loss of activity Collectively, these data show that two-step purified MC99-3FLAG is an appropriate substrate to study tissue-derived c-secretase activity that is inhibited by a specific c-secretase inhibitor and is detergent concentration-sensitive Mammalian expression and proteolytic processing of SPC99-3FLAG An alternative approach to monitoring c-secretase activity was developed using a novel mammalian expression vector The SPA4CT sequence, which corresponds to the C-terminal 99 amino acids of human APP fused to the APP signal peptide [18], was ligated into a C-terminal 3-FLAG repeat expression vector and the resulting construct, SPC99-3FLAG (Fig 3A), was used for expression of c-secretase substrate in mammalian cells Anti-FLAG western blot analysis of FEBS Journal 272 (2005) 5544–5557 ª 2005 FEBS COS-7 cells transfected with SPC99-3FLAG (COS-7SPC99-3FLAG cells) shows the expected cleavage product by signal peptidase (Fig 3B and C) Previous data with the SPA4CT construct showed that signal peptidase cleavage resulted in a 101 amino acid protein with the amino acids LE fused to the N terminus of Ab [19], thus the protein was named C101-3FLAG C-terminal fragments produced from COS7-SPC993FLAG cells were analysed by co-electrophoresis of cell lysates with c-3FLAG or e-3FLAG standards Anti-FLAG western blot analysis shows that the CTF from COS7-SPC99-3FLAG cells has an electrophoretic mobility indistinguishable from that of e-3FLAG standard (Fig 3B, lane 1) but a slightly faster mobility than the c-3FLAG standard (Fig 3C, lane 1) Using C101-3FLAG, c-3FLAG and e-3FLAG standards as molecular mass markers, the FLAG-reactive band migrating below C101-3FLAG is calculated to be a protein resulting from the expected a-secretase cleavage [20] (see Experimental procedures) Lastly, longer exposures allowed the detection of a protein with a calculated molecular mass of 11.1 kDa migrating between a- and c-3FLAG standard proteins that may correspond to a minor a-secretase cleavage product [1] (Fig 3C) Therefore, SPC99-3FLAG is expressed in mammalian cells as a C101-3FLAG protein that undergoes the expected processing by a- and c-cleavages to produce a CTF corresponding to cleavage at the e-site 5547 Characterization of in vitro c-secretase activity D E Hoke et al A D B E C F Fig Expression of SPC99-3FLAG in COS-7 cells and detection of c-secretase activity in whole-cell and cell-free assays (A) Schematic of the SPC99-3FLAG protein (B) Anti-FLAG Western blot analysis of extracts from COS7-SPC99-3FLAG cells Lane 1, spiked with e-3FLAG standard; Lane 2, lysate sample alone Note that the intensity of the e-CTF-3FLAG band was greater in lane spiked with e-3FLAG standard (C) A similar experiment to that in (B) was performed except that lane is a sample spiked with c-3FLAG standard indicated by the arrow marked ‘c std’ Lane 2, lysate sample alone Note that a separation between c-3FLAG standard and e-CTF-3FLAG was achieved in lane An uncharacterized anti-FLAG immunoreactive protein migrating between the a and c peptides was detected on long exposures (indicated by the arrow on the right of panel C) (D) In vitro assay with CHAPSO-solubilized c-secretase from COS7-SPC99-3FLAG cells 1% CHAPSO extracts from COS7-SPC99-3FLAG cells were diluted to 0.5% and incubated at 37 or °C for 16 h with dimethylsulfoxide or the c-secretase inhibitor L-685,458 at the concentrations indicated The reactions were analysed by anti-FLAG Western blot Note that activity was abolished in a dose-dependent manner upon addition of L-685,458 (E) Sensitivity of COS-7-SPC99-3FLAG soluble c-secretase assay in 0.5% CHAPSO: 4, 2, or lg soluble c-secretase was diluted to 0.5% CHAPSO, incubated at 37 or °C and analysed by anti-FLAG Western blot Note that e-CTF production was detected from lg of membrane extract (F) COS7-SPC99-3FLAG soluble c-secretase was diluted to 0.25% CHAPSO and 2, 1, and 0.5 lg of extract tested for activity Note that faint activity was seen with lg extract In vitro c-secretase assay with COS7-SPC993FLAG solubilized membranes To complement our MC99-3FLAG based in vitro c-secretase assays, an in vitro assay using COS7SPC99-3FLAG CHAPSO extracts was developed The substrate in this assay is synthesized, processed, and trafficked in the cell and would theoretically be presented to the c-secretase complex in a more native state than E coli-derived substrate Anti-FLAG western blot analysis was used to monitor the generation of e-CTF Upon 16 h incubation of a 0.5% CHAPSOsolubilized membrane preparation at 37 °C, a robust e-CTF signal was detected while a similar signal was not observed upon incubation at °C (Fig 3D) This activity was inhibited in a dose-dependent manner by the c-secretase inhibitor L-685,458 with a similar potency as seen for the MC99-3FLAG-based assay (Fig 2A) and previous reports [17] The sensitivity of the COS-7 SPC99-3FLAG c-secretase assay was explored in relation to extract amount and CHAPSO content e-C-terminal fragment production could be detected in a dose-dependent fashion with as little as lg of cell membrane extract diluted in 0.5% 5548 CHAPSO (Fig 3E) Using COS7-SPC99-3FLAG extracts diluted in 0.25% CHAPSO, dose-dependent c-secretase activity was detected but the sensitivity was increased, allowing activity to be detected from lg of extract (Fig 3F) Collectively these data show that COS7-SPC99-3FLAG extracts can be used to monitor c-secretase activity and that this activity is sensitive to CHAPSO concentration PS molecular mass and c-secretase activity from COS7-SPC99-3FLAG cells is altered by size exclusion chromatography in a CHAPSO concentration-dependent fashion Much controversy exists within the literature concerning the molecular mass of c-secretase complexes and activity Since our assays measure activity without the need of overexpressing the c-secretase complex components and are highly sensitive under diluting conditions, we set out to determine the molecular mass of activity by size exclusion chromatography Unlike blue native PAGE, this method allows the simultaneous determination of c-secretase complexes size and activity A 1% CHAPSO extract from COS-7-SPC99-3FLAG cells FEBS Journal 272 (2005) 5544–5557 ª 2005 FEBS D E Hoke et al was diluted to 0.5% CHAPSO and chromatographed on a Superose column equilibrated with 0.5% CHAPSO This CHAPSO concentration was chosen as it is compatible with c-secretase activity (as shown in Fig 3E) and it results in a lesser dilution of sample than the previously published 0.25% CHAPSO concentration [21] Because the c-secretase complex components were endogenous, only low amounts were present such that detection by western blot analysis was limited to our most sensitive assay for PS1 N-terminal fragment (NTF) Fractions were analysed for the presence of C101-3FLAG and PS1 NTF and the signals quantified by image densitometry (Fig 4A) A broad peak of C101-3FLAG immunoreactivity was found in fractions corresponding to 440–25 kDa while PS1 NTF was detected in a 669-kDa peak These data indicate that very little of the substrate co-fractionates with c-secretase complexes Gamma-secretase activity from 0.5% CHAPSO columns was tested by pooling fractions, adding phospholipids, and incubating at 37 or °C, followed by immunoprecipitation with anti-FLAG agarose No generation of e-CTF was observed in any of the pooled fractions We hypothesized that not enough substrate cofractionated with PS complexes to allow the production of a detectable signal However, when exogenous MC99-3FLAG substrate and phospholipid was added to fractions, activity was not detected Thus, substrate limitation is not the reason that PS1 complexes of this size range were unable to sustain robust c-secretase activity Original reports on the size of PS1 and c-secretase activity by size exclusion chromatography showed that both eluted at the void volume [21] However, these authors used 0.25% CHAPSO during column chromatography As we observed that the CHAPSO concentration had an effect on c-secretase activity in unseparated materials, we repeated the size exclusion experiment in the presence of 0.25% CHAPSO Immunoblots for PS1 NTF showed elution at the void volume (Fig 4B), a result in contrast to the 669-kDa peak obtained with chromatography in presence of 0.5% CHAPSO Because most of C101-3FLAG immunoreactivity was again found in fractions between 440 and 25 kDa, separate from the fractions containing PS1, c-secretase activity acting upon transfected C101-3FLAG was not tested Rather, fractions were tested by adding exogenous MC99-3FLAG and phospholipids (Fig 4C) Under these conditions, the fractions eluting at the void volume were able to produce a strong e-CTF signal upon incubation at 37 °C It was noted that activity did not directly correlate to the amount of PS1-NTF present in these FEBS Journal 272 (2005) 5544–5557 ª 2005 FEBS Characterization of in vitro c-secretase activity A B C Fig Superose size fractionation of COS7-SPC99-3FLAG soluble c-secretase (A) Sixty-seven micrograms of 1% CHAPSO cell membrane extract was diluted to 0.5% CHAPSO and loaded onto a Superose column equilibrated in 0.5% CHAPSO Arrows at the top indicate elution of molecular mass standards PS1 NTF fractionates in 669-kDa fractions while C101-3FLAG fractionates between 440 and 25 kDa (B) Twenty micrograms of CHAPSO extract from COS-7 SPC99-3FLAG cells was diluted to 0.25% CHAPSO and applied to a Superose column equilibrated in 0.25% CHAPSO Presenilin-1 NTF immunoreactivity was detected from column fractions with a peak near the void volume C101-3FLAG immunoreactivity was detected as a peak between 440 and 25 kDa (C) Fractions from (B) were assayed for c-secretase activity using exogenous MC99-3FLAG substrate and phospholipids as described These reactions were incubated at 37 or °C for 17 h and analysed by anti-FLAG Western blot Lanes containing the e-3FLAG standard are indicated by ‘e’ Gamma-secretase activity was detected in void volume fractions only Note that e-3FLAG production per fraction pool did not correlate directly to the amount of presenilin-1 NTF present in those fractions pooled fractions These results indicate that endogenous c-secretase activity from COS-7 cells is associated with a CHAPSO concentration-sensitve complex 5549 Characterization of in vitro c-secretase activity in the megaDalton range and suggest that only a subset of PS-containing c-secretase complexes are enzymatically active Gamma-secretase activity from guinea pig brain membrane is altered by size exclusion chromatography in a CHAPSO concentrationdependent fashion To extend these results, 1% CHAPSO membrane extracts of guinea pig brain were subjected to Superose chromatography in the presence of 0.25% or 0.5% CHAPSO and assayed for c-secretase activity on exogenous MC99-3FLAG substrate A mgỈmL)1 1% CHAPSO extract was diluted to 500 lgỈmL)1 in 0.25% CHAPSO and 400 lL (200 lg) loaded onto the column Gamma-secretase activity was detected in high D E Hoke et al molecular mass fractions in duplicate column runs (Fig 5A) The c-secretase complex components Nct, PS1, and PS2 were likewise found primarily in high molecular mass fractions but not exactly overlapping with c-secretase activity (Fig 5B) Aph1a, Aph1b, and Pen2 could not be detected in any fraction due to sample dilution during chromatography Similarly, a mgỈmL)1 1% CHAPSO extract was diluted to mgỈmL)1 in 0.5% CHAPSO and 400 lL (400 lg) loaded onto a Superose column Gamma-secretase activity was not detected in any fraction (Fig 5C), confirming that column chromatography in the presence of 0.5% CHAPSO resulted in a loss of c-secretase activity Interestingly, when Nct, PS1, and PS2 immunoreactivity was tested in these 0.5% CHAPSO fractions it was found that a significant amount of these proteins were present in high molecular mass fractions A C B D Fig Size fractionation of guinea pig brain soluble c-secretase by Superose column chromatography (A) Guinea pig brain soluble c-secretase (200 lg) was diluted to 0.25% CHAPSO, and chromatographed on a Superose column equilibrated in 0.25% CHAPSO One-ml fractions were collected and aliquots assayed for c-secretase activity using exogenous MC99-3FLAG substrate and phospholipids Gamma-secretase activity was measured by densitometry as described Gamma-secretase activity was detected mainly in fractions 10 and 11 (B) Fractions from the 0.25% CHAPSO column were analysed for the presence of mature (mat) and immature (imm) nicastrin (Nct), PS1 NTF, PS1 CTF, and PS2 by western blot These proteins were present mainly in fractions 11 and 12 Note that c-secretase complex component levels did not directly correlate to the amount c-secretase activity (C) Guinea pig brain soluble c-secretase (400 lg) was diluted to 0.5% CHAPSO and separated in 0.5% CHAPSO No activity was detected in any fraction tested (D) Fractions from the 0.5% CHAPSO column were analysed for c-secretase complex components by western blot Note increases in Nct and PS2 immunoreactivity migrating between 440 and 25 kDa in 0.5% CHAPSO fractions compared to 0.25% CHAPSO fractions These results (A–D) are typical of duplicate column runs 5550 FEBS Journal 272 (2005) 5544–5557 ª 2005 FEBS D E Hoke et al Characterization of in vitro c-secretase activity (Fig 5D) However, in contrast to 0.25% CHAPSO chromatography, equivalent amounts of PS2 and mature and immature Nct could be found in low molecular mass fractions Thus 0.5% CHAPSO during chromatography abolishes c-secretase activity and causes a subset of both Nct isoforms and PS2 to migrate in lower molecular mass fractions Dimethylsulfoxide can modulate detection of COS7-SPC99-3FLAG in vitro c-secretase activity While performing control reactions for inhibitor experiments, a two- to fivefold increase in the detection of products arising from in vitro c-secretase activity was observed when adding 2.5% v ⁄ v dimethylsulfoxide (the inhibitor solvent) in the assay This observation was complemented by performing a dimethylsulfoxide dose–response in the COS7-SPC99-3FLAG c-secretase assay in 0.5% CHAPSO Epsilon-CTF detection was enhanced fivefold by 2.5%, enhanced slightly by 5%, and decreased by 10% dimethylsulfoxide when compared to non-dimethylsulfoxide control reactions (Fig 6) These data show that dimethylsulfoxide can enhance or decrease detection of c-secretase activity depending on the concentration used Zinc treatment of COS7-SPC99-3FLAG CHAPSO extracts causes C101-3FLAG to elute at a high molecular mass Zinc binding to Ab has been shown to promote Ab oligomerization [22–25] Since a functioning zinc-binding domain may be present in the Ab sequence of C99, we hypothesized that zinc may affect the oligomerization state of C101-3FLAG Therefore, the molecular mass of C101-3FLAG before and after zinc treatment was determined by size exclusion chromatography (Fig 7) Without the addition of zinc, C101-3FLAG eluted as a peak in the 67–43-kDa molecular mass range After treatment with ZnCl2, C101-3FLAG eluted Fig Effects of dimethylsulfoxide on the detection of in vitro c-secretase cleavage of C101-3FLAG CHAPSO-solubilized (0.5%) c-secretase from COS7-SPC99-3FLAG cells was incubated in the absence or presence of dimethylsulfoxide at the concentrations indicated Adding 2.5% dimethylsulfoxide significantly increased e-CTF signal compared to 0% and 10% dimethylsulfoxide reactions FEBS Journal 272 (2005) 5544–5557 ª 2005 FEBS Fig C101-3FLAG size fractionated by Superose 12 chromatography in the presence of zinc shows an increased molecular mass CHAPSO extracts of COS7-SPC99-3FLAG cells were incubated in buffer with or without 234 lM Zn before loading onto a column equilibrated in the same buffer with or without zinc The fractions were analysed by anti-FLAG western blot for C101-3FLAG immunoreactivity with the resulting C101-3FLAG signal quantified by image densitometry This data (y-axis) was plotted according to fraction number (x-axis) The elution points for blue dextran (void), BSA (67 kDa), ovalbumin (43 kDa), and chymotrypsinogen (25 kDa) are indicated by arrows as a high molecular mass peak corresponding to the void volume of this column These data show that zinc can alter the apparent molecular mass of an APPderived c-secretase substrate Zinc inhibits c-secretase activity in COS7-SPC993FLAG and MC99-3FLAG based assays We hypothesized that zinc-induced substrate oligomerization may affect its ability to be cleaved Therefore, the effect of zinc on the two in vitro c-secretase assays was determined Firstly CHAPSO extracts from COS7SPC99-3FLAG membranes were incubated with ZnCl2 (Fig 8A) This inhibited c-secretase substrate cleavage with a 50% inhibitory concentration (IC50) of lm Zn To verify these findings, they were repeated in a second assay system using MC99-3FLAG as a substrate for guinea pig brain membrane-derived c-secretase activity The counterion dependence for zinc was tested by using ZnCl2 (Fig 8B) and ZnSO4 (Fig 8C) Regardless of the counterion, zinc inhibited cleavage of MC99-3FLAG with comparable IC50 values of 22 and lm Zn Using two assay systems, these results show that zinc can inhibit in vitro c-secretase cleavage of an APP substrate Discussion The amyloid hypothesis of AD states that low molecular mass oligomers of Ab initiate cellular toxicity leading to memory loss and dementia [26,27] Thus blocking Ab formation by inhibiting c-secretase is a 5551 Characterization of in vitro c-secretase activity A B D E Hoke et al C Fig Zinc inhibits in vitro c-secretase activity (A) 0.5% CHAPSO-solubilized c-secretase from COS7-SPC99-3FLAG cells was incubated with ZnCl2 This resulted in a dose-dependent inhibition of activity (B, C) CHAPSO-solubilized (0.5%) c-secretase from guinea pig brain acting upon the MC99-3FLAG substrate was incubated with ZnCl2 (B), and ZnSO4 (C) to show a dose-dependent decrease in c-secretase activity with increasing zinc content The quantitated data is shown in graphical form under each panel strategy for the prevention of AD An initial step in discovering c-secretase inhibitors is the development of assays that monitor c-secretase activity This paper describes two novel in vitro c-secretase assays During the development of these assays we identified an Ab-like NTF and e-like CTF from extracts of MC993FLAG-transformed E coli Secondly, we show that detergent concentration can affect the apparent size of the c-secretase complex components and affect c-secretase activity which correlates to a subset of PS complexes Thirdly, dimethylsulfoxide can modulate the detection of in vitro c-secretase activity Lastly we show that zinc causes a change in the apparent molecular mass of a c-secretase substrate and inhibits c-secretase cleavage Using a purification protocol that minimized the E coli-derived e-CTF-like contamination, purified MC99-3FLAG was used to detect c-secretase activity from rodent brains An alternative in vitro assay was developed by solubilizing membranes from COS-7 cells transfected with the SPC99-3FLAG construct Previous studies have shown that MC99 tagged with a single FLAG motif forms SDS-insoluble aggregates [28] We also found higher molecular mass species of MC99-3FLAG and C101-3FLAG after SDS ⁄ PAGE Therefore, like Ab it would appear that C99 is inherently aggregating When comparing the molecular mass of E coli-to COS-7-derived substrates, significant differences are seen While nondenaturing size exclusion chromatography of MC99-transformed E coli extracts yields a void volume molecular mass determination, C101-3FLAG is found mainly in 67–43-kDa 5552 fractions This shows that E coli and mammalian cells have different mechanisms to control the aggregation states of these substrates and supports our original hypothesis that mammalian cellular factors enable endogenous proteins to be presented to the c-secretase complex in a different state than exogenous substrate When Superose size exclusion chromatography was used to separate COS7-SPC99-3FLAG and guinea pig brain membrane extracts in the presence of 0.5% CHAPSO, c-secretase activity was not detected despite numerous attempts and the addition of exogenous phospholipids Calculations allowing for a 50% theoretical loss during chromatography, and the fact that only an aliquot of each fraction was assayed still placed the theoretical yield well within the detection limits of our assay which showed that activity could be detected with lg of extract regardless of enzyme source, dilution, or CHAPSO concentration Therefore, the reason for a lack of c-secretase activity cannot be attributed to low assay sensitivity Size-separation of c-secretase using 0.25% CHAPSO as the column buffer allowed detection of c-secretase activity despite using less starting material than for 0.5% CHAPSO columns Analysis of fractions from COS7-SPC99-3FLAG separations showed a shift for PS1 NTF to low molecular mass fractions after chromatography in the presence of 0.5% CHAPSO as compared to elution at the void volume of the column in the presence of 0.25% CHAPSO Fractionation of guinea pig brain membrane extracts did not show as dramatic a decrease in the c-secretase complex molecular mass upon 0.5% CHAPSO chromatography as all of the FEBS Journal 272 (2005) 5544–5557 ª 2005 FEBS D E Hoke et al components examined were present in a high molecular mass complex However, a partial decomposition of the complex had occurred since equivalent amounts of mature and immature Nct and PS2 were detected in high and low molecular mass fractions of the 0.5% CHAPSO separations when compared to the recovery of these proteins predominantly in high molecular mass fractions during 0.25% CHAPSO separation Collectively these data show that increasing detergent upon column chromatography can partially dissociate PS1 and PS2 c-secretase complexes While preparing this manuscript, a report by Wrigley et al [29] showed that overexpressed c-secretase complex components yielded c-secretase activity that was abolished during chromatography on a Superose 6HR column in the presence of 0.5% CHAPSO However they found that activity could be restored by adding exogenous phospholipids While we were not able to restore activity with the addition of phospholipids, these results show that by keeping the CHAPSO concentration at 0.25%, significant activity can be recovered from size exclusion chromatography separations When comparing c-secretase activity from 0.25% CHAPSO-separated fractions to the presence of PS1 NTF in those fractions, we noted that activity and PS levels did not directly correlate The greatest amount of activity was always present in the highest molecular mass fractions before PS levels had peaked These data suggest that a subset of PS involved in the highest complexed state yields significant activity as has been suggested by other methods previously [30] and by inhibitor binding assays [31,32] A restrospective analysis of the work by Li et al [21] also indicates an imperfect relationship between activity and PS NTF ⁄ CTF levels The successful recovery of native activity after size-exclusion chromatography, described in this work, is an important step in identifying the factors that enable c-secretase cleavage in these highest molecular mass fractions Our data show that detection of e-CTFs from in vitro c-secretase activity can be increased two- to fivefold by the addition of 2.5% dimethylsulfoxide Three hypotheses for this effect can be made Firstly, dimethylsulfoxide can alter c-secretase enzyme kinetics through its ability to interact with the phospholipid bilayer [33–36] Secondly, dimethylsulfoxide could stabilize the c-secretase complex making it act longer without altering the rate of proteolysis As dimethylsulfoxide affects the phase behaviour of bilayers it probably affects the c-secretase complex which is composed of at least 18 transmembrane domains and its interaction with transmembrane substrates This is supported by our work and by other studies showing its activity is highly sensitive to factors that FEBS Journal 272 (2005) 5544–5557 ª 2005 FEBS Characterization of in vitro c-secretase activity modulate membrane structure and stability, including detergent type [21], detergent concentration [21,28,37], and phospholipid content [12,28] However, until a detailed kinetic analysis is made we cannot exclude a third hypothesis that the endproduct of proteolysis is stabilized by dimethylsulfoxide in a concentration-dependent fashion Our results indicate that in vitro c-secretase cleavage of APP substrates is inhibited by zinc and that zinc increases the apparent molecular mass of C1013FLAG as determined by size exclusion chromatography Residues 6–28 within Ab constitute a domain that binds metal ions such as zinc, copper, and iron and mediates Ab aggregation ([23] reviewed [38]) This is the first report suggesting that this metal binding domain is functional within APP C99 causing oligomerization with the biochemical consequence of inhibiting c-secretase cleavage This mechanism is supported by correlations between the metal-dependent IC50 for c-secretase inhibition and the affinity constants for Ab interaction with metals Firstly, the 7–22 lm IC50 for zinc inhibition of c-secretase activity correlates with the reported 5.2-lm dissociation constant for a low affinity Ab interaction with zinc [23] Secondly, just as zinc is the most potent metal mediating Ab aggregation, we found that c-secretase inhibition by zinc was approximately 10 times more potent than copper (D.E.H., unpublished data) These results suggest that the Ab metal-binding site within APP C99 causes oligomerization to a noncleavable state An alternate explanation for the effect of zinc and copper inhibition is an interaction between metals and phospholipid bilayers Zinc has been shown to be the most potent metal in dehydrating lipid bilayers with copper being the second most potent [39,40] As water molecules are necessary for most proteoytic processes, zinc and copper modulation of the hydration state of lipid bilayers may control c-secretase activity regardless of substrate Future experiments with c-secretase substrates that not bind metals will clarify the mechanism by which zinc and copper inhibit in vitro c-secretase activity A universal characteristic of AD pathology is the post-mortem detection of Ab plaques, thus confirming the pathological relevance of c-secretase cleavage of APP Since only a small subset of AD cases are linked to mutant PS or APP proteins, it has been hypothesized that disease modifying genes and environmental factors account for the common pathology of Ab plaque formation in sporadic cases Here we have shown that dimethylsulfoxide, and detergent concentrations alter in vitro c-secretase activity While these experimental manipulations could not be compared to environmental factors they show that agents known to 5553 Characterization of in vitro c-secretase activity modify phospholipid bilayers can modulate in vitro c-secretase activity positively or negatively Likewise, high cholesterol levels have been shown to increase the risk of AD [41] and some reports have suggested that this occurs through modulation of c-secretase activity by changes in membrane structure [29] A large body of literature has suggested that zinc and copper levels in the brain could be environmental-derived factors in AD pathogenesis [22,42] Recently, several mouse models have confirmed a key role of zinc [43–46] and copper [47,48] in Ab plaque formation The finding that in vitro c-secretase cleavage of APP is inhibited by physiologically relevant concentrations of zinc places metal-mediated modulation of this activity as a potential mechanism for the metal-mediated modification of Ab plaque formation and Ab biogenesis Experimental procedures Construction of expression vectors for SPC99-3FLAG, MC99-3FLAG, c-3FLAG standard and e-3FLAG standard The following primer pairs were used to PCR amplify the SPA4CT sequence [18]: forward, 5¢-CCCAAGCTTGGGT GCCCCGCGCAGGGTCGCG-3¢; reverse, 5¢-GGGGGG GATCCGTTCTGCATCTGCTC-3¢ This product was then ligated into the HindIII ⁄ BamHI site of p3XFLAG-CMV-14 and the resulting vector named SPC99-3FLAG The following primer pairs were used to amplify the C99-3FLAG sequence from SPC99-3FLAG: forward, 5¢-GGGGGGCC ATGGATGCAGAATTCCGAC-3¢; reverse, 5¢-GGGGGG AAGCTTTTACTTGTCATCGTCATCC-3¢ (reverse 3FLAG HindIII) This product was ligated into the NcoI ⁄ HindIII site of pTrcHisA (Invitrogen, Carlsbad, CA, USA) resulting in a plasmid named MC99-3FLAG The following primers were used to amplify the 40-3FLAG sequence from the SPC99-3FLAG vector: forward, 5¢-GGGGGGCCAT GGCGACAGTGATCGTC-3¢; reverse, 3FLAG HindIII creating the plasmid c-3FLAG standard Finally, the primer pairs forward, 5¢-GGGGGGCCATGGTGATGCTGA AGAAGAACAG-3¢ and reverse 3FLAG HindIII were used to generate the plasmid e-3FLAG standard D E Hoke et al h This mixture was then centrifuged at 18 000 g and the supernatant transferred to a separate tube This supernatant was brought up to 10% glycerol (v ⁄ v) and loaded onto a Superdex-75 (Pharmacia, Fairfield, CT, USA) column equilibrated with 0.5% (v ⁄ v) Triton X-100 in NaCl ⁄ Pi Fractions were analysed by anti-FLAG western blot analysis Fractions rich in MC99-3FLAG but depleted in lower molecular mass cleavage products were pooled These pooled fractions were then applied to an anti-FLAG, M2 agarose column (Sigma, St Louis, MO, USA), washed with Hepes buffer + 0.5% (v ⁄ v) Triton X-100 and eluted with mL 0.1 m glycine, 0.15 m NaCl, 20% (v ⁄ v) glycerol, pH 4.0 into 80 lL m Tris ⁄ HCl pH 9.0 The 2-mL eluate was used in exogenous substrate c-secretase assays Preparation of c- and e-3FLAG proteins Escherichia coli transformed with the c- and e-3FLAG vectors were prepared as above Pellets were sonicated in lysis buffer [1% (v ⁄ v) Triton X-100, 1% (v ⁄ v) NP40, mm MgCl2, mm EDTA, 50 mm Tris ⁄ HCl pH 7.5] with : 100 protease inhibitor cocktail solution and centrifuged at 3000 g The supernatant was purified by anti-FLAG affinity chromatography as above Preparation of soluble c-secretase from PS1 A246E transgenic mouse brain, guinea pig brain, and COS7-SPC99-3FLAG cells Whole brains minus the cerebellum were minced with a razor blade in Hepes buffer plus 1% protease inhibitor cocktail (Sigma) COS7-SPC99-3FLAG cell pellets stored at )80 °C were thawed and suspended in Hepes buffer This suspension was then subjected to repeated passages through successively smaller needles down to 25 G The homogenate was centrifuged at 3000 g for 20 °C and the supernatant subjected to a 100 000 g centrifugation for h at °C The pellet was then homogenized in carbonate buffer (0.1 m Na2CO3 pH 11.2) and centrifuged at 100 000 g for h °C The final carbonate-washed pellet was washed twice with Hepes buffer before resuspension in Hepes buffer + 1% (v ⁄ v) CHAPSO and mixing end-over-end at °C for h The suspension was centrifuged at 18 000 g for at room temperature and the supernatant, named ‘soluble c-secretase’, was aliquotted and stored at )80 °C Preparation of MC99-3FLAG Escherichia coli was grown, induced, and harvested as in [49] Eshcherichia coli pellets were then sonicated in Hepes buffer (50 mm Hepes, mm MgCl2, mm CaCl2, 0.15 m KCl) +1% (w ⁄ v) protease inhibitor cocktail and centrifuged at 100 000 g to create a soluble and membrane fraction The 100 000 g pellet was homogenized in Hepes buffer + 1% (v ⁄ v) CHAPSO by repeated passage through a 25-G needle and incubated with end-over-end rocking for 5554 MC99-3FLAG c-secretase assay with soluble c-secretase from mouse brain extracts, guinea pig brain membrane extracts and column fractions MC99-3FLAG was added to soluble c-secretase or sizefractionated soluble c-secretase at a : 60 dilution Experiments in which the CHAPSO content was not indicated FEBS Journal 272 (2005) 5544–5557 ª 2005 FEBS D E Hoke et al were performed in 0.5% CHAPSO Dimethylsulfoxide or L685,458 in dimethylsulfoxide, were added in equivalent volumes to make control and inhibitor reactions Metal inhibition assays were performed by incubating soluble c-secretase activity from guinea pig brain membrane extracts with equal volumes of glycine buffer (0.1 m glycine pH 7.0), or ZnCl2 ⁄ ZnSO4 dissolved in glycine buffer to make control and experimental reactions that were incubated at 37 °C for h 3-sn-Phosphatidylethanolamine from bovine brain and l-a-phosphatidylcholine from egg yolk were added to fractions from sizing columns at a final concentration of 2.5 lgỈmL)1 each as described previously [28] Reactions were then subjected to 37 or °C incubation for 12–18 h Reactions were stopped by adding SDS sample buffer and then analysed by anti-FLAG western blot with M2 monoclonal antibody (Sigma) Cell lines and transfections COS-7 cells were tranfected by lipofectamine 2000 according to the manufacturer’s protocol (Invitrogen) Stable COS7-SPC99-3FLAG cell lines were established by selection with 300 lgỈmL)1 geneticin G418 Determining the molecular mass of FLAG immunoreactivities from COS7-SPC99-3FLAG cells COS7-SPC99-3FLAG cell extracts were separated by electrophoresis using tricine gels [50] and Anti-FLAG western blot analysis was performed An electrophoretic mobility vs molecular mass graph was prepared using C1013FLAG, c-3FLAG and e-3FLAG standards with the resulting line having a correlation coefficient of 0.998 This line was used to predict the molecular mass of the CTF migrating faster than C101-3FLAG as an a-CTF within 27 Da of the calculated molecular mass Finally the same line was used to predict the molecular mass of a third FLAG-reactive protein between alpha and c-3FLAG proteins as 11.1 kDa In vitro c-secretase assays with COS7-SPC993FLAG cells Assays were performed by thawing soluble c-secretase, diluting to 0.5% or 0.25% (v ⁄ v) CHAPSO in Hepes buffer and incubating at °C or 37 °C for 2–16 h Experiments in which the CHAPSO content was not indicated were performed in 0.5% CHAPSO Inhibitor assays were incubated with equivalent volumes of dimethylsulfoxide or L-685,458 diluted in dimethylsulfoxide Metal inhibition assays were performed by incubation with equal volumes of Hepes buffer or ZnCl2 dissolved in Hepes buffer to make control ⁄ experimental reactions that were incubated at 37 °C FEBS Journal 272 (2005) 5544–5557 ª 2005 FEBS Characterization of in vitro c-secretase activity for h The assays were stopped by adding SDS sample buffer and the reactions were separated on tricine gels [50] Size exclusion chromatography A · 30 cm column was packed with Superose resin and calibrated with blue dextran (void volume), ferritin (880kDa dimer eluted at the void volume and 440-kDa monomer), thyroglobulin (669 kDa), and chymotrypsinogen A (25 kDa) A · 30-cm column was packed with Superose 12 resin and calibrated with blue dextran (void volume), BSA (67 kDa), ovalbumin (43 kDa), and chymotrypsinogen (25 kDa) All solutions were filtered through a 0.2-lm filter prior to the addition of CHAPSO CHAPSO solutions were then filtered through Whatman paper Glycerol was added to soluble c-secretase (10% glycerol, v ⁄ v) plus Hepes buffer making the final CHAPSO concentration 0.5% or 0.25% (v ⁄ v) The column was equilibrated with at least five column volumes of buffer with the same CHAPSO ⁄ Hepes composition as the sample Zinc or control columns were equilibrated with 0.25% (v ⁄ v) CHAPSO in Hepes buffer with or without 487 lm ZnCl2 Finally, the sample was applied to the column, separated at a flow rate of 0.1 mLỈmin)1, and fractions collected Quantitation of c-secretase activity and inhibition by zinc nih image software (version 1.63) was used to quantify the density of e-CTF immunoreactivity in 37 °C, °C, and zinc-treated reactions Acknowledgements We thank L D Canterford and K Uaesoontrachoon for technical assistance and Dr M Shearman for providing L-685,458 inhibitor We would also like to thank Drs D A Caruso, K J Barnham, A I Bush, and R A Cherny for helpful discussion The graphics expertise of J C Hoke is also appreciated This work was supported by a Ruth L Kirschstein NRSA individual fellowship from the United States NIH-NIA to D.E.H (AG05887) and by the Australian NHMRC (program grant 208978) References Weidemann A, Eggert S, Reinhard FB, Vogel M, Paliga 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