In vitro c-secretase assay with COS7-SPC99-3FLAG solubilized membranes To complement our MC99-3FLAG based in vitro c-secretase assays, an in vitro assay using COS7-SPC99-3FLAG CHAPSO ext
Trang 1amyloid 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 Genevie`ve M Evin
Department of Pathology, The University of Melbourne and the Mental Health Research Institute, Parkville, Victoria, Australia
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
ecto-domain 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 1 and 2 (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 muta-tions have been found in the APP gene, the majority linked to early onset familial AD occur in the
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 4 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, ante-rior pharynx defective, nicastrin, and presenilin enhancer-2 cleaves the C-terminal 99-amino acid fragment of the amyloid precursor protein intra-membranously 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, frag-ments 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 size-fractionated 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 know-ledge of the complexes and biochemical factors needed for c-secretase activity and suggest a mechanism by which zinc dysregulation may contrib-ute to Alzheimer’s disease pathogenesis
Abbreviations
AD, Alzheimer’s disease; APP, amyloid precursor protein; CTF, C-terminal fragment; NTF, N-terminal fragment; PS, presenilin.
Trang 2transmembrane region near c and e cleavage sites (as
reviewed in [5]) Gamma-secretase activity is attributed
to an integral membrane complex of the four
trans-membrane proteins: PS, nicastrin (Nct), anterior
pha-rynx-defective, and presenilin enhancer 2 (as reviewed
in [6])
In vitro c-secretase assays have been essential in
elu-cidating the mechanism of inhibitors [7–9], the
struc-ture 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
mem-brane proteins are not necessary for activity as
car-bonate 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
des-cribe 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
secretase substrate and acts as an inhibitor of
c-secretase 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-ter-minal 99 amino acids of APP and a C-terC-ter-minal triple
FLAG tag The resulting protein was named
MC99-3FLAG (Fig 1A) Escherichia coli expression vectors
encoding 3FLAG-tagged APP-CTFs mimicking
prod-ucts 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)
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 immunoreact-ive peptides The major product migrated at 18 kDa and was also detected by anti-Ab antibody, WO2 From its apparent molecular mass and its immunore-activity, it can be concluded that it corresponds to MC99-3FLAG (Fig 1B and C) There were also sev-eral higher molecular mass and degraded species iden-tified 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 west-ern 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 varia-tions 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 immuno-reactivity was detected in lysates of mock-transformed cells (Fig 1E) Collectively, these data indicate that upon expression or during purification, a small frac-tion of MC99-3FLAG is degraded into multiple spe-cies 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 prep-arations 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 vol-ume 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 tes-ted for cleavage by c-secretase from PS1A246E trans-genic mouse brain [16] (Fig 2A) prepared by solubilization of carbonate-washed membranes with
Trang 31%
[3-[(3-cholamidopropyl)dimethylammonio-]-2-hyd-roxy-1-propanesulfonate] (CHAPSO) Upon
incuba-tion at 37C, generation of an 9 kDa CTF was
detected by western blotting with FLAG
anti-body 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)
Assay sensitivity was tested by varying the enzyme amount, enzyme dilution, and CHAPSO concentra-tion For this experiment, c-secretase activity was pre-pared by extracting carbonate-washed guinea pig brain membranes with 1% CHAPSO A 2 mgÆmL)1 extract was diluted to obtain a final concentration of 0.5% CHAPSO, and dilutions were made in 0.5% CHAPSO
to 20 lgÆmL)1 Incubation of these dilutions with sub-strate showed c-secretase activity to be enzyme dose-dependent, and that signal was detectable using the
40 lgÆmL)1 dilution of extract Therefore, as little as
1 lg of membrane extract was sufficient to obtain a signal (Fig 2B) Secondly, the 2 mgÆmL)1 extract was
A
Fig 1 Escherichia coli produces peptides similar to Ab and an e-CTF when trans-formed with MC99-3FLAG (A) Schematic of proteins (B) Lysate from MC99-3FLAG-expressing E coli, c and e standards were separated by SDS ⁄ PAGE and western blot-ted for FLAG immunoreactivity MC99-3FLAG 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 trans-formed with the MC99-3FLAG expression vector was probed with monoclonal anti-body 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) Anti-FLAG western blot analysis of lysate from MC99-3FLAG-transformed E coli alone or spiked with different amounts of the e-3FLAG standard An uncharacterized anti-FLAG 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 sim-ilar experiment to that shown in (D) was performed by spiking MC99-3FLAG-trans-formed E coli lysate with c-3FLAG stand-ard This standard migrated slower than the
E coli product Mock-transformed E coli had no background anti-FLAG immuno-reactivity.
Trang 4diluted to 0.25% CHAPSO and subsequently diluted
in 0.25% CHAPSO to 40 lgÆmL)1 In contrast to the
0.5% CHAPSO dilution, 0.25% dilution did not show
a dose-dependent relation of enzyme amount to
prod-uct formed (Fig 2C) Rather the highest concentration
and amount showed little activity while the least
con-centrated sample (1.2 lg of 40 lgÆmL)1) showed the
highest activity and greater than the corresponding
dilution in 0.5% CHAPSO Perhaps this 0.25%
con-centration was not enough to keep high concon-centrations
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
con-centration-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
cor-responds 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
COS-7 cells transfected with SPC99-3FLAG (COS-7-SPC99-3FLAG 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-SPC99-3FLAG 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 cleav-age [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-cleav-ages to produce a CTF corresponding to cleavage at the e-site
C
Fig 2 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 4 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 gen-eration of e-CTF-3FLAG (e) signal upon incubation at 37 C over background 4 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 exo-genous 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 4 C for 15 h The reactions were analysed by anti-FLAG Western blot Gamma-secretase activity was detected using as little as 1 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.
Trang 5In vitro c-secretase assay with
COS7-SPC99-3FLAG solubilized membranes
To complement our MC99-3FLAG based in vitro
c-secretase assays, an in vitro assay using
COS7-SPC99-3FLAG CHAPSO extracts was developed The
substrate in this assay is synthesized, processed, and
trafficked in the cell and would theoretically be
presen-ted 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%
CHAPSO-solubilized membrane preparation at 37C, a robust
e-CTF signal was detected while a similar signal was
not observed upon incubation at 4C (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 2 lg of cell membrane extract diluted in 0.5%
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 1 lg of extract (Fig 3F) Collectively these data show that COS7-SPC99-3FLAG extracts can be used to mon-itor c-secretase activity and that this activity is sensi-tive 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 concern-ing the molecular mass of c-secretase complexes and activity Since our assays measure activity without the need of overexpressing the c-secretase complex compo-nents 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 determin-ation of c-secretase complexes size and activity A 1% CHAPSO extract from COS-7-SPC99-3FLAG cells
A
Fig 3 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 1 spiked with e-3FLAG standard (C) A similar experiment to that in (B) was performed except that lane 1 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 1.
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 4 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 1 lg soluble c-secretase was diluted to 0.5% CHAPSO, incubated at 37 or 4 C and analysed by anti-FLAG Western blot Note that e-CTF production was detected from 2 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 1 lg extract.
Trang 6was diluted to 0.5% CHAPSO and chromatographed
on a Superose 6 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
con-centration [21] Because the c-secretase complex
com-ponents 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 4C, 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
Immu-noblots for PS1 NTF showed elution at the void
vol-ume (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
trans-fected 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
pro-duce a strong e-CTF signal upon incubation at
37C It was noted that activity did not directly
cor-relate to the amount of PS1-NTF present in these
pooled fractions These results indicate that endo-genous c-secretase activity from COS-7 cells is associ-ated with a CHAPSO concentration-sensitve complex
A
B
C
Fig 4 Superose 6 size fractionation of COS7-SPC99-3FLAG soluble c-secretase (A) Sixty-seven micrograms of 1% CHAPSO cell mem-brane extract was diluted to 0.5% CHAPSO and loaded onto a Superose 6 column equilibrated in 0.5% CHAPSO Arrows at the top indicate elution of molecular mass standards PS1 NTF fractio-nates in 669-kDa fractions while C101-3FLAG fractiofractio-nates 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 6 column equilibrated in 0.25% CHAPSO Presenilin-1 NTF immunoreactivity was detected from column frac-tions with a peak near the void volume C101-3FLAG immuno-reactivity was detected as a peak between 440 and 25 kDa (C) Fractions from (B) were assayed for c-secretase activity using exo-genous MC99-3FLAG substrate and phospholipids as described These reactions were incubated at 37 or 4 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.
Trang 7in the megaDalton range and suggest that only a
sub-set of PS-containing c-secretase complexes are
enzy-matically active
Gamma-secretase activity from guinea pig brain
membrane is altered by size exclusion
chromatography in a CHAPSO
concentration-dependent fashion
To extend these results, 1% CHAPSO membrane
extracts of guinea pig brain were subjected to Superose
6 chromatography in the presence of 0.25% or 0.5%
CHAPSO and assayed for c-secretase activity on
exo-genous MC99-3FLAG substrate A 2 mgÆmL)1 1%
CHAPSO extract was diluted to 500 lgÆmL)1in 0.25%
CHAPSO and 400 lL (200 lg) loaded onto the
col-umn Gamma-secretase activity was detected in high
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
2 mgÆmL)1 1% CHAPSO extract was diluted to
1 mgÆmL)1 in 0.5% CHAPSO and 400 lL (400 lg) loaded onto a Superose 6 column Gamma-secretase activity was not detected in any fraction (Fig 5C), confirming that column chromatography in the pres-ence of 0.5% CHAPSO resulted in a loss of c-secretase activity Interestingly, when Nct, PS1, and PS2 immu-noreactivity was tested in these 0.5% CHAPSO frac-tions it was found that a significant amount of these proteins were present in high molecular mass fractions
A
C
Fig 5 Size fractionation of guinea pig brain soluble c-secretase by Superose 6 column chromatography (A) Guinea pig brain soluble c-secretase (200 lg) was diluted to 0.25% CHAPSO, and chromatographed on a Superose 6 column equilibrated in 0.25% CHAPSO One-ml fractions were collected and aliquots assayed for c-secretase activity using exogenous MC99-3FLAG substrate and phospho-lipids Gamma-secretase activity was measured by densitometry as described Gamma-secretase activity was detected mainly in frac-tions 10 and 11 (B) Fracfrac-tions 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-secret-ase (400 lg) was diluted to 0.5% CHAPSO and separated in 0.5% CHAPSO No activity was detected in any fraction tested (D) Frac-tions 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.
Trang 8(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
cau-ses 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-bind-ing domain may be present in the Ab sequence of C99,
we hypothesized that zinc may affect the
oligomeriza-tion 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
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 APP-derived c-secretase substrate
Zinc inhibits c-secretase activity in COS7-SPC99-3FLAG and MC99-COS7-SPC99-3FLAG based assays
We hypothesized that zinc-induced substrate oligomeri-zation 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 COS7-SPC99-3FLAG membranes were incubated with ZnCl2 (Fig 8A) This inhibited c-secretase substrate cleavage with a 50% inhibitory concentration (IC50) of 7 lm
Zn To verify these findings, they were repeated in a second assay system using MC99-3FLAG as a sub-strate for guinea pig brain membrane-derived c-secret-ase 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 9 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 molecu-lar mass oligomers of Ab initiate cellumolecu-lar toxicity lead-ing to memory loss and dementia [26,27] Thus blocking Ab formation by inhibiting c-secretase is a
Fig 7 C101-3FLAG size fractionated by Superose 12 chromato-graphy 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 l M 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 immuno-reactivity 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.
Fig 6 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.
Trang 9strategy 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
MC99-3FLAG-transformed E coli Secondly, we show that
detergent concentration can affect the apparent size of
the c-secretase complex components and affect
c-secre-tase activity which correlates to a subset of PS
com-plexes 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
Previ-ous studies have shown that MC99 tagged with a
sin-gle 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
inher-ently aggregating When comparing the molecular
mass of E coli-to COS-7-derived substrates, significant
differences are seen While nondenaturing size
exclu-sion chromatography of MC99-transformed E coli
extracts yields a void volume molecular mass
determin-ation, C101-3FLAG is found mainly in 67–43-kDa
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 6 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% theor-etical 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 2 lg of extract regardless of enzyme source, dilution, or CHAPSO concentration There-fore, the reason for a lack of c-secretase activity can-not 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 chroma-tography in the presence of 0.5% CHAPSO as com-pared 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
Fig 8 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 ZnCl 2 (B), and ZnSO 4 (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.
Trang 10components 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
pro-teins predominantly in high molecular mass fractions
during 0.25% CHAPSO separation Collectively these
data show that increasing detergent upon column
chro-matography can partially dissociate PS1 and PS2
c-secr-etase 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%
CHA-PSO 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
com-plexed state yields significant activity as has been
sugges-ted 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
rela-tionship 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
kinet-ics through its ability to interact with the
phospho-lipid bilayer [33–36] Secondly, dimethylsulfoxide
could stabilize the c-secretase complex making it act
longer without altering the rate of proteolysis As
di-methylsulfoxide affects the phase behaviour of
bilay-ers 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
modulate membrane structure and stability, inclu-ding detergent type [21], detergent concentration [21,28,37], and phospholipid content [12,28] How-ever, until a detailed kinetic analysis is made we can-not 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 C101-3FLAG as determined by size exclusion chromato-graphy 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 oligo-merization with the biochemical consequence of inhib-iting 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 aggrega-tion, 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 cop-per modulation of the hydration state of lipid bilayers may control c-secretase activity regardless of substrate Future experiments with c-secretase substrates that do 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 hypothes-ized that disease modifying genes and environmental factors account for the common pathology of Ab pla-que formation in sporadic cases Here we have shown that dimethylsulfoxide, and detergent concentrations alter in vitro c-secretase activity While these experi-mental manipulations could not be compared to envir-onmental factors they do show that agents known to