From the Institute of Gerontology and Department of
Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109 and the ¶ Department of Neurology, University of Michigan Medical
Center, Ann Arbor, Michigan 48109 and Veterans Affairs Medical Center
Geriatric Research, Education, and Clinical Center,
Ann Arbor, Michigan 48105
Received for publication, September 26, 2000, and in revised form, November 16, 2000
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ABSTRACT |
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Amyloid (A Evidence continues to accumulate supporting the hypothesis that
amyloid plaques in the brain have a causative role in the generation of
Alzheimer's disease (for review, see Ref. 1). Increased brain levels
of amyloid peptide and cognitive decline are strongly correlated (2).
Amyloid plaques largely consist of peptides of 40 (A Several laboratories have now cloned an enzyme that cleaves APP and
APPSw at the Before the cloning of the aspartic protease, BACE, cellular studies
using a serine protease inhibitor, 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF), suggested that it blocked A By developing an ELISA specific for the soluble Antibodies and Protease Inhibitors--
The mouse monoclonal
antibodies referred to in Fig. 1A as 22C11 and
BIOSOURCE were obtained from Roche Molecular
Biochemicals and BIOSOURCE International
(monoclonal antibody P2-1), respectively. The mouse monoclonal antibody
6E10, which recognizes an epitope in the first 17 amino acids of the
A
The peptide aldehyde, MG132, was dissolved in dimethyl sulfoxide
(Me2SO) at a concentration of 10 mM (Peptides
International). The proteasomal inhibitor,
clasto-lactacystin Cell Culture and Transfection--
The Chinese hamster ovary
cell line, CHOK1, and the HEK293 cell line used for transient
transfections were obtained from the American Type Culture Collection.
The cell lines stably expressing the 695 isoforms of either APPSw
(CHOAPPSw) or APP wild type were a generous gift from Taraneh Haske
(Pfizer Pharmaceuticals, Ann Arbor, MI). All CHO cell lines were grown
in DMEM supplemented with 10% heat-inactivated fetal calf serum,
glutamine, nonessential amino acids, and
penicillin/streptomycin/fungizone as described (19). Experiments
involving transient expression of APPSw or APP were conducted using
cDNAs encoding for the 751 isoforms that were cloned into the
pCDNA3 mammalian expression vector (Invitrogen, Inc.).
Transfections of HEK293 cells grown on 60-mm plates were conducted
using LipofectAMINE as described by the Life Technologies, Inc. Forty
hours following transfection, cells were metabolically labeled or lysed
for immunoblot analysis as described below.
Metabolic and Pulse-Chase Labeling--
Cells stably or
transiently expressing APP or APPSw were preincubated in
methionine/cysteine-free medium for 15 min prior to labeling. Cells
were metabolically labeled by incubating in 2 ml of medium containing
[35S]methionine and [35S]cysteine
(Tran35S-label; ICN Pharmaceuticals) at 50 µCi/ml for
1 h. In pulse-chase studies, cells were preincubated in
methionine/cysteine-free medium for 15 min and then pulsed for 12 min
with Tran35S-label (100 µCi/ml). Cells were then washed
and incubated in complete medium containing excess methionine and
cysteine for the chase times shown. After labeling and chase were
complete, the conditioned medium was collected and cells washed in PBS. Cells were lysed in 1 ml of lysis buffer (0.5% Nonidet P-40, 0.5% deoxycholate in 50 mM Tris, 150 mM NaCl, and 5 mM EDTA, pH 8.0) and insoluble cell debris removed by
centrifugation as described (19). The resulting cleared supernatant of
the cell lysate was then subjected to further analysis.
Immunoprecipitation, Immunoblotting, and Gel
Electrophoresis--
Full-length APP and APPSw were isolated from the
cell lysate supernatants by immunoprecipitation using the 945 rabbit
antisera to the carboxyl terminus of APP. Except where noted, lysates
were incubated with 4 µl of 945 antisera for 90 min at 4 °C and
protein-antibody complexes were isolated by incubation with protein
A-Sepharose for 30 min at 4 °C. Intracellular APPSw
Isolated proteins were resolved by SDS-polyacrylamide gel
electrophoresis (PAGE) using an 8% separating gel. Radiolabeled proteins in SDS gels were detected by fluorography using Amplify (Amersham Pharmacia Biotech). Immunoblot analysis of isolated proteins
was conducted essentially as described (19). Briefly, immunoprecipitated proteins were resolved by SDS-PAGE and transferred to PROTRAN (Schleicher & Schuell). Membranes were subsequently blocked
in gelatin wash buffer (0.1% gelatin, 15 mM Tris, pH 7.5, 130 mM NaCl, 1 mM EDTA, and 0.1% Triton
X-100). Membranes were subsequently incubated with mouse monoclonal
antibody 22C11 to detect the amino-terminal end of APP molecules from
cell lysates or conditioned medium. Membranes were alternatively
incubated with 6E10 to detect APPSw Full-length APP and APPSw
For the full-length APP ELISA, the protein concentration of the cell
lysate supernatant was determined using the BCA protein assay (Pierce).
Ten micrograms of protein from the cell lysates were aliquoted into
each well. The total volume in the well was adjusted to 100 µl with
PBS, and samples were incubated overnight at 4 °C. On the following
day, the samples were incubated an additional 1 h with constant
shaking. The wells were then washed four times with PBS/T and then
incubated with 100 µl of diluted detector antibody (the mouse
monoclonal antibody, 8E5, at 0.25 µg/ml except where noted). All
antibodies used in the ELISA were diluted using a solution of 10%
SuperBlock and 90% PBS/T. The detector antibody was incubated with
each sample for 4 h with constant shaking. The wells were again
washed four times with PBS/T and subsequently incubated for 1 h
with rabbit anti-mouse IgG conjugated to horseradish peroxidase
(diluted 1:4000; Southern Biotechnology Associates, Inc.). Following
three washes with PBS/T and two washes with PBS alone, 100 µl of
3,3',5,5'-tetramethylbenzidine (Pierce) solution was added to each
well. The reaction was stopped by the addition of an equal volume of 2 M sulfuric acid. The relative amount of full-length APP in
the sample was then quantified colorimetrically at 450 nm. The levels
of secreted APPSw Characterization of Polyclonal Antibodies to APP and
APPSw
As others have demonstrated, the Characterization of ELISAs for Detection of Full-length APP and the
Soluble
The 8E5 and BIOSOURCE mouse monoclonal antibodies
were also evaluated as detector antibodies in the 931 ELISA to measure
secreted APPSw Intracellular APPSw
Taken together, the results described above indicated that
iAPPSw The APPSw
The highly specific proteasomal inhibitor, lactacystin, increases
APP
Intriguingly, peptide aldehyde protease inhibitors, which are capable
of inhibiting both the proteasome and cysteine proteases, increase A
The continued increase in APPSw
MG132 had little effect on secretion of APPSw In previous studies, peptide aldehydes such as MG132 were thought
to inhibit A Our results on MG132-induced inhibition of The 931 antibody, described here for the first time, specifically
recognized APPSw Although BACE is an aspartic protease, 1 mM AEBSF (a serine
protease inhibitor) reportedly blocks secretion of A The reported effects of peptide aldehyde protease inhibitors on A Although BACE activity in CHO cells was the highest of all non-neuronal
cells, it was still significantly less than that observed for neuronal
cell cultures (7). Therefore, the anti-APPSw) peptides found aggregated into
plaques in Alzheimer's disease are derived from the sequential
cleavage of the amyloid precursor protein (APP) first by
- and then
by
-secretases. Peptide aldehydes, which inhibit cysteine proteases
and proteasomes, reportedly block A
peptide secretion by interfering
with
-secretase cleavage. Using a novel, specific, and sensitive
enzyme-linked immunosorbent assay for the
-secretase-cleaved
fragment of the Swedish mutant of APP (APPSw), we determined that the
peptide aldehyde, MG132, prevented
-secretase cleavage. This block
in
-secretase cleavage was not observed with
clasto-lactacystin
-lactone and thus, cannot be
attributed to proteasomal inhibition. MG132 inhibition of
-secretase
cleavage was compared with the serine protease inhibitor,
4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF). AEBSF
inhibition of
-secretase cleavage was immediate and did not affect
-secretase cleavage. With MG132, inhibition was delayed and it
decreased secretion of
-cleaved APPSw as well. Furthermore, MG132
treatment impaired maturation of full-length APPSw. Both inhibited
intracellular formation of the
-cleaved product. These results
suggest that peptide aldehydes such as MG132 have multiple effects on
the maturation and processing of APP. We conclude that the
MG132-induced decrease in
-secretase cleavage of APPSw is due to a
block in maturation. This is sufficient to explain the previously
reported peptide aldehyde-induced decrease in A
peptide secretion.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
40)1 and 42 (A
42)
amino acids in length that are derived by the enzymatic processing of a
type I transmembrane protein called amyloid precursor protein (APP).
Two enzymatic cleavages of APP are necessary to produce amyloid
peptides. First,
-secretase cleaves APP to create the amino-terminal
end of the peptide. A double mutation (K651N/M652L; 751 isoform
numbering) just amino-terminal to this
-secretase cleavage site has
been identified in a Swedish pedigree of familial Alzheimer's disease
(3). This double mutation of APP, known as the "Swedish" mutation
(APPSw), elevates intracellular and secreted levels of A
peptide
from 6- to 8-fold (4). This appears to be a consequence of increased
cleavage of APPSw by
-secretase compared with wild type APP.
Following
-secretase cleavage,
-secretase subsequently cleaves
the COOH-terminal membrane-bound fragment (CTF) of APP within the
transmembrane sequence to release the A
peptide. Thus, inhibitors
that specifically block the cleavage of APP by these secretases have
enormous therapeutic potential.
-secretase site (5-7). Referred to as BACE (
-site APP cleaving
enzyme), this enzyme is a member of a unique family of
transmembrane aspartic proteases. A second related protein designated
ASP1 or BACE2 has also been identified (6, 8, 9). However, the
expression pattern of BACE2 in the brain appears to exclude it from
playing a major role in Alzheimer's disease (9). The mature, fully
glycosylated form of BACE has a half-life in the cell of greater than
9 h (10). BACE appears to localize to the Golgi apparatus (5).
Despite about 40% amino acid similarity between BACE and pepsin
proteases (9), the cysteine residues in BACE involved in intramolecular
disulfide bonds are not conserved with other pepsin family members
(10). Such fundamental structural differences may explain why
-secretase is insensitive to pepstatin, a specific inhibitor of
pepsin proteases (7). The search for such a specific inhibitor of
-secretase cleavage of APP as a possible treatment for Alzheimer's
disease has intensified with the discovery of BACE.
peptide generation by directly inhibiting
-secretase activity (11). There is
little information published about other potential specific inhibitors
of
-secretase. Several studies have shown that peptide aldehyde
protease inhibitors affect the secretion of A
peptides (12-14). Of
these peptide aldehydes, MG132 was one of the most potent inhibitors of
A
peptide secretion (15). Studies focusing specifically on the
secretion of A
40 and A
42 revealed peptide aldehydes to have a
complex effect on APP processing. Curiously, at low concentrations,
these peptide aldehydes produced an increase in A
40 and A
42
peptide secretion, whereas, at higher concentrations, a decrease in
A
peptide secretion was observed (14-17). The increase in A
peptide secretion at low concentrations of peptide aldehydes has been
postulated to result from inhibition of degradation of the CTFs
generated by BACE cleavage, making more of them available for
-secretase cleavage (17). The inhibition of A
peptide secretion
with higher concentrations of peptide aldehydes is attributed to an
impairment of
-secretase cleavage of the CTFs (12, 13, 17, 18).
-secretase-cleaved
amino-terminal fragment of APPSw (APPSw
; see Fig. 1A), we
now report that the peptide aldehyde, MG132, prevents
-secretase cleavage of APPSw in a concentration-dependent manner.
Furthermore, MG132 is not inhibiting secretion of APPSw
into the
medium since it blocked intracellular production of APPSw
as well.
MG132 is compared with AEBSF and the specific proteasomal inhibitor,
clasto-lactacystin
-lactone, on
-secretase cleavage.
MG132 impairs maturation, blocking
-secretase cleavage of APPSw in
the late Golgi apparatus. This offers an alternative explanation as to
how higher concentrations of a peptide aldehyde can decrease A
peptide secretion.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
peptide, was obtained from Senetek, Inc. The anti-APP
COOH-terminal rabbit polyclonal antibody was obtained from Chemicon
International, Inc., and the mouse monoclonal antibody to the amino
terminus of APP, LN27, was purchased from Zymed Laboratories
Inc. The mouse monoclonal antibody, 8E5, was a generous gift from
Dr. Dale Schenk (Elan Pharmaceuticals). The rabbit polyclonal antibody,
945, was raised against a synthetic peptide corresponding to the last
19 amino acid residues of the carboxyl terminus of APP
(CMQQNGYENPTYKFFEQMQN) that was cross-linked to keyhole limpet
hemocyanin via an amino-terminal cysteine. The polyclonal antibodies,
931 and 932, were raised against a synthetic peptide corresponding to
19 amino acid residues (CRPGSGLTNIKTEEISEVNL) just amino-terminal to
the
-secretase cleavage site of APPSw that was similarly
cross-linked to keyhole limpet hemocyanin via an amino-terminal cysteine.
-lactone (Calbiochem), was also
dissolved in Me2SO at 2 mM. Me2SO
was used in all experiments as a vehicle control. The serine protease
inhibitor AEBSF (Sigma) was dissolved in sterile water at 0.2 M.
was similarly
isolated from cell lysates using 4 µl of 931 rabbit antisera. The 931 antisera was also used to isolate secreted APPSw
from 0.5 ml of
conditioned medium that had Nonidet P-40 and deoxycholate added to a
final concentration of 0.5%. Immunoprecipitations using the mouse
monoclonal antibodies, 8E5 or 6E10, were conducted as described above
except that protein-antibody complexes were isolated using protein
G-agarose (Roche Molecular Biochemicals).
in conditioned medium. Membranes
were subsequently washed and incubated with a sheep anti-mouse IgG antibody conjugated to horseradish peroxidase as described by the
manufacturer (Amersham Pharmacia Biotech). The membranes were again
washed and signals detected by chemiluminescence using the ECL system
(Amersham Pharmacia Biotech).
Sandwich ELISAs--
The 945 and
931 antisera were used as capture antibodies for the full-length APP
and APPSw
sandwich ELISAs, respectively. Each was first
affinity-purified against the appropriate peptide that had been
cross-linked to CNBr-activated Sepharose 4B (Amersham Pharmacia
Biotech) following the manufacturer's instructions. The anti-APP
COOH-terminal antibody, 945, was affinity-purified with the same
20-amino acid synthetic peptide used above to inoculate rabbits. The
931 antibody was affinity-purified using a synthetic peptide identical
to the last seven amino acids of the neoepitope of the
-secretase-cleaved APPSw soluble fragment (CEIESVNL). The 945 and
931 antibodies that bound to the immobilized peptide were eluted using
ActiSep Elution Medium (Sterogene). The eluted antibodies were desalted
using a PD-10 column (Amersham Pharmacia Biotech) as directed by the
manufacturers. The affinity-purified capture antibodies (945 and 931)
were diluted to 1.2 µg/ml in PBS and 100 µl added per well to a
96-well Nunc-Immuno Maxisorp plate (Nalge Nunc International). After
incubation to allow binding of antibody, SuperBlock (Pierce) was added
to each well to block nonspecific binding sites. The wells were
repeatedly rinsed and then stored at 4 °C in PBS, 0.05% Tween 20 (PBS/T) until ready for use.
were assayed in the exact same way except that
plates coated with affinity-purified 931 antibody were used to capture
the
-secretase-cleaved protein and just 25 µl of conditioned
medium was loaded per well. Each experiment was repeated at least three
times, and the indicated values are averages of triplicate
measurements ± S.D.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
--
As shown in Fig. 1,
-secretase cleavage of APPSw generates a large soluble
amino-terminal fragment (APPSw
) that is secreted into the medium and
a CTF that is subsequently cleaved by
-secretase to derive the A
peptide. Two novel rabbit polyclonal antisera were generated to perform
the experiments described below. The binding sites of these and other
antibodies are also shown in Fig. 1. The first polyclonal antisera
(945) was raised to the last 20 amino acids of the APP carboxyl
terminus. The specificity of the anti-COOH-terminal antibody, 945, is
shown in Fig. 2A. CHO cells
stably expressing APPSw (CHOAPPSw) were metabolically labeled for
1 h with Tran35S-label. Following cell lysis, equal
amounts of supernatant were incubated with 1, 2, or 4 µl of 945 antiserum or with 4 µl of preimmune serum followed by protein
A-Sepharose. The immunoprecipitated APPSw was compared with that
obtained using the Chemicon anti-COOH-terminal antibody by SDS-PAGE.
The resulting autoradiograph shows that 945 specifically
immunoprecipitates the N-glycosylated immature (I) and completely glycosylated mature (M) forms
of APPSw. The second antisera specifically recognizes only the carboxyl
terminus of the soluble amino-terminal fragment (APPSw
) created when
-secretase cleaves APPSw just COOH-terminal to Leu652 (751 numbering). Results shown in Fig. 2B demonstrate that
antisera raised in two rabbits (931 and 932) against the 20-amino acid
sequence just amino-terminal to the
-secretase cleavage site are
capable of immunoprecipitating a soluble APPSw fragment from the
conditioned medium of CHOAPPSw cells. Culture medium conditioned for
24 h by CHOAPPSw cells was divided into equal aliquots and
incubated with preimmune sera from rabbit 931, 931 antisera, or 932 antisera. The mouse monoclonal antibody, 8E5, which recognizes an
epitope in the lumenal region of APP (Fig. 1), served as a positive
control. The immunoprecipitates were resolved by SDS-PAGE and the APPSw
amino-terminal fragments were identified by Western blot analysis using
the mouse monoclonal antibody 22C11. Both 931 and 932 antisera, but not
preimmune sera, immunoprecipitated a specific APP soluble fragment from
the conditioned media. The 931 antisera appeared to have a higher titer
than 932. Thus, all subsequent experiments utilized only 931.
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Fig. 1.
A schematic illustration showing full-length
APPSw and products of -secretase
cleavage. The region of APP that gives rise to A
peptide is
shown in gray, and the region to become the soluble APPSw
fragment is indicated by arrows. The sites of
-,
-,
and
-secretase cleavages are shown on the full-length protein. Also
shown are the relative locations of epitopes for antibodies used in
this study. TMD refers to the transmembrane domain.
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Fig. 2.
Characterization of novel polyclonal
antibodies. A, the ability of 945 to immunoprecipitate
full-length APPSw was compared with the Chemicon rabbit polyclonal
antibody to the carboxyl terminus of APP. CHOAPPSw cells were
metabolically labeled with [35S]cysteine and
[35S]methionine for 1 h. Cells were lysed and equal
aliquots of cleared supernatant incubated with the indicated amounts of
polyclonal antisera or with 4 µl of preimmune serum followed by
protein A-Sepharose. M and I, respectively, refer
to the mature, fully glycosylated and immature,
N-glycosylated forms of APPSw. B, immunoblot
analysis of proteins specifically immunoprecipitated from conditioned
medium of CHOAPPSw cells. Two newly derived rabbit polyclonal antisera
(931 and 932) were used to immunoprecipitate APP soluble fragments from
conditioned medium collected from CHOAPPSw cells. This was compared
with APP fragments immunoprecipitated by the 8E5 antibody by
immunoblotting with 22C11, which recognizes both APPSw and APPSw
fragments. C, immunoblot analysis indicates that the rabbit
polyclonal antisera 931 specifically immunoprecipitates only APPSw
from conditioned media. Conditioned media collected from HEK293 cells
transiently transfected with either pCDAPP or pCDAPPSw were incubated
with either 8E5 or 931 and protein-antibody complexes immunoabsorbed
using protein G- or protein A-agarose, respectively. Duplicate samples
of immunoprecipitated proteins were resolved by SDS-PAGE and
transferred to nitrocellulose. Panel 1, one set
of samples was immunoblotted with the 22C11 mouse monoclonal antibody
to detect secreted APP
, APPSw
, APP
, and APPSw
.
Panel 2, a duplicate set of immunoprecipitated
samples was immunoblotted for APP
and APPSw
using the mouse
monoclonal antibody, 6E10. D, two ELISAs described under
"Experimental Procedures" were derived to measure APPSw
in
conditioned media and full-length APP or APPSw in cell lysates.
Affinity-purified 945 antibody was used to capture full-length APPSw
molecules from the lysate of CHOAPPSw cells. Three different mouse
monoclonal antibodies that recognize epitopes in the lumenal region of
human APP were screened as detector antibodies. Lysates of CHO cells
expressing only endogenous hamster APP served as controls for
specificity. E, affinity-purified 931 antibody was used to
capture secreted APPSw
molecules from the conditioned media of
CHOAPPSw cells. CHO cell conditioned medium served as a negative
control. Both of the anti-APP lumenal domain antibodies, 8E5 and
BIOSOURCE, were effective detector antibodies.
Addition of the CEIESVNL peptide to the CHOAPPSw conditioned medium
prevented detection of secreted APPSw
.
- and
-cleaved soluble fragments
are difficult to electrophoretically resolve from one another (20, 21).
Therefore, it was unclear from this Western blot analysis of
conditioned medium whether the soluble APPSw fragment
immunoprecipitated by 931 was limited to secreted APPSw
or also
included
-secretase-cleaved APPSw (APPSw
). Furthermore, it was
not known if 931 could immunoprecipitate wild type APP
and APP
.
Consequently, HEK293 cells were transiently transfected with either
pCDAPP or pCDAPPSw. Medium that had been conditioned for 36 h by
these transiently transfected cells was collected and soluble APP
fragments were immunoprecipitated with 931 or the mouse monoclonal
antibody 8E5. Since the epitope recognized by 8E5 is amino-terminal to
the
-secretase cleavage site, it is capable of immunoprecipitating
APP
, APP
, APPSw
, and APPSw
. Immunoprecipitates were
resolved by SDS-PAGE and immunoblotted using the mouse monoclonal
antibody, 22C11 to detect all forms of soluble APP and APPSw (see Fig.
1). As expected, 8E5 was able to immunoprecipitate both secreted APP
and APPSw from the conditioned media (Fig. 2C,
panel 1). However, 931 only immunoprecipitated soluble APPSw amino-terminal fragments from the conditioned medium of
cells transiently transfected with pCDAPPSw and did not recognize wild
type secreted APP fragments. A set of immunoprecipitations from
conditioned medium identical to that conducted in Fig. 2C (panel 1) was immunoblotted with the mouse
monoclonal antibody 6E10, which detects only secreted APP and APPSw
soluble fragments that have been cleaved by
-secretase. Although
both APP
and APPSw
were detected in the 8E5 immunoprecipitates,
neither form was immunoprecipitated using the 931 antibody (Fig.
2C, panel 2). Taken together, these
results demonstrate that the 931 antibody specifically recognizes only
the neoepitope derived with
-secretase cleavage of APPSw. It does
not cross-react with full-length APPSw (compare Fig. 4, A
and C, chase at 0 min), soluble APP
, APP
, or
APPSw
.
-Secretase Cleaved APPSw Amino-terminal Fragment--
Since
931 specifically recognized the neoepitope of
-secretase-cleaved
APPSw in conditioned medium, it was used to create an enzyme-linked
immunosorbent assay (ELISA) to quantitatively measure the amount of
secreted APPSw
. A similar ELISA was also developed to measure the
amount of full-length APP or APPSw present in cell lysates using 945. To identify the best detector antibody to use in the ELISA, the
affinity-purified 945 was coated on 96-well plates to capture
full-length APP from cell lysates of either CHO cells or CHO cells
stably expressing APPSw. After rinsing, triplicate wells were incubated
with no detector, 8E5 (0.25 µg/ml), BIOSOURCE
(0.25 µg/ml), or LN27 (0.5 µg/ml) mouse monoclonal antibodies and
developed as described under "Experimental Procedures." The signal
level observed using CHO cell lysates with these detector antibodies
did not significantly differ from background, demonstrating the
specificity of the ELISA for the stably expressed, human APPSw (Fig.
2D). Full-length APPSw was specifically detected in CHOAPPSw lysates with 8E5 and the BIOSOURCE mouse
monoclonal antibodies showing the greatest sensitivity. A total protein
concentration of 10 µg of cell lysate was found to be optimal to
detect full-length APPSw (data not shown).
. Both 8E5 and BIOSOURCE
antibodies were sensitive detectors of APPSw
captured in wells
coated with affinity-purified 931 (Fig. 2E). The addition of
the eight-amino acid synthetic peptide corresponding to the carboxyl
terminus of APPSw
to the conditioned medium from CHOAPPSw cells
blocked detection of the
-cleaved product. When using 6E10 as the
detector antibody, no signal above background was observed (data not
shown). This indicated that no APPSw
was captured in the ELISA by
the affinity-purified 931. Together, these results demonstrate the high
specificity of this novel ELISA for APPSw
.
Is Detected by Pulse-Chase
Analysis--
Experiments were next conducted to determine whether 931 recognized intracellular APPSw
. Intracellular APPSw
was
detectable in the cell lysate as a slightly diffuse band migrating just
above a nonspecific band (Fig.
3A, panel
2). This nonspecific 35S-labeled band was
detected in both CHOAPP and CHOAPPSw cell lysates (data not shown).
Therefore, an additional experiment was conducted to verify that this
protein was not recognized by antibodies specific for the APPSw
neoepitope. CHOAPPSw cells were pulse-labeled with Tran35S-label for 12 min and chased for 45 min as described
under "Experimental Procedures." Conditioned media and cell lysates
were isolated and divided in half. An excess of a synthetic peptide
with a sequence corresponding to the last eight amino acids of the
neoepitope (EISEVKNL) in APPSw
was added to one set of samples.
Secreted and intracellular APPSw
were then immunoisolated using 931 from conditioned media and cell lysates, respectively. Full-length APPSw was subsequently immunoprecipitated from the same cell lysates using 945. Full-length APPSw was still immunoprecipitated in the presence of the neoepitope peptide, indicating that the peptide was not
causing a general block in the immunoisolation of proteins (Fig.
3A, panel 1). However, the synthetic
neoepitope peptide blocked immunoprecipitation of secreted APPSw
(Fig. 3A, panel 2) and intracellular
APPSw
(Fig. 3A, panel 3) using 931 antibody. In contrast, the nonspecific band was still detectable in the presence of synthetic peptide. Brefeldin A (BFA) treatment of HEK293
cells to block transport of proteins beyond the Golgi apparatus inhibited
-secretase cleavage of APPSw (20, 22). Thus, a block in
maturation of APPSw by BFA does not permit access to BACE in the late
Golgi. We sought to confirm these findings using the 931 antisera.
CHOAPPSw cells were metabolically labeled with Tran35S-label for 1 h in the presence or absence of 20 µg/ml brefeldin A. Labeled conditioned medium was collected and cells
lysed as above. Intracellular and secreted APPSw
were isolated by
immunoprecipitation using the 931 antisera from cell lysates and
conditioned media, respectively. Full-length APPSw was subsequently
immunoisolated from cell lysates using the 945 antibody. As expected,
BFA treatment blocked the appearance of APPSw
in the conditioned
medium (Fig. 3B, panel 3). Completely
glycosylated, mature full-length APPSw was not detected in BFA-treated
cell lysates indicating that maturation was blocked (panel
1). Similar to earlier reports (20, 22), our studies found
that intracellular APPSw
was not detected in CHOAPPSw cells treated
with BFA, yet the nonspecific band was still detectable (Fig.
3B, panel 2). Thus, blocking
maturation of APPSw prevents it from being cleaved by
-secretase.
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Fig. 3.
Immunoprecipitation of radiolabeled
intracellular and secreted APPSw is blocked by
a synthetic peptide of the Swedish mutant and brefeldin A
treatment. A, CHOAPPSw cells were pulse labeled as
described and chased for 45 min in complete DMEM. Conditioned media and
cell lysates were isolated and divided in half. 931 was used to
immunoprecipitate secreted and intracellular APPSw
in the presence
or absence of excess synthetic peptide corresponding in sequence to the
carboxyl terminus of APPSw
(CEIESVNL). 1, the excess
peptide did not interfere with immunoprecipitation of full-length APPSw
with 945. 2, the CEIESVNL peptide blocked
immunoprecipitation of iAPPSw
(indicated by an
arrow), but not the nonspecific protein just below it.
3, secreted APPSw
was not immunoprecipitated by 931 when
the CEIESVNL peptide was added to the conditioned medium. B,
CHOAPPSw cells were metabolically labeled for 1 h with
Tran35S-label in the presence (+) or absence (
) of 20 µg/ml brefeldin A (BFA). Conditioned medium was collected and cell
lysates isolated as described. Full-length APPSw was immunoprecipitated
with 945 antisera (panel 1). Intracellular
(indicated by arrow in panel 2) and
secreted (panel 3) APPSw
were
immunoprecipitated from cell lysate and conditioned media,
respectively, with 931 antisera.
is detectable in the lysates of radiolabeled
CHOAPPSw cells. Consequently, a pulse-chase study was conducted to
evaluate the time course of APPSw
production and secretion by
CHOAPPSw cells. Cells were pulsed for 12 min with
Tran35S-label and subsequently chased in complete medium
containing excess methionine and cysteine for 0-90 min. At each
indicated chase time, conditioned medium was collected and the
PBS-washed cells were lysed. Intracellular APPSw
was
immunoprecipitated from the cleared supernatants using 931. Full-length
APPSw was subsequently immunoisolated from the supernatants using 945. Secreted APPSw
was immunoprecipitated from the conditioned media
with 931. These radiolabeled proteins were resolved by SDS-PAGE, and the resulting autoradiographs are shown in Fig.
4. A 12-min pulse was sufficient to label
N-glycosylated APPSw (I in Fig. 4C)
but not fully glycosylated, mature APPSw (M). Mature APPSw
was detectable after 7.5 min of chase and peaked after 25-35 min.
After a 90-min chase, no mature APPSw was detectable and only a trace
of immature protein was still present. Note that a large portion of
APPSw fails to chase into the fully mature form over the chase period, but instead appears to remain as incompletely glycosylated APPSw. This
may represent misfolded protein that is retained in the ER for eventual
degradation by the proteasome in these APPSw-overexpressing cells. Fig.
4A shows that iAPPSw
was readily detectable
after chasing for about 25 min and corresponded with the appearance of
the completely glycosylated, mature APPSw. The level of
iAPPSw
reached a maximum at 35-45 min and was barely
detectable after 90 min of chase. Thus, as expected, the appearance of
iAPPSw
preceded the secretion of APPSw
into the medium
(Fig. 4B). Only a trace of intracellular APPSw
was
detected after 35 min, while secreted APPSw
continued to accumulate
after chasing for 90 min.
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Fig. 4.
Pulse-chase analysis of intracellular
APPSw production and secretion from CHOAPPSw
cells. Cells were pulse-labeled for 12 min with
Tran35S-label and chased for the indicated times in minutes
in complete DMEM with excess unlabeled methionine and cysteine as
described under "Experimental Procedures." A,
iAPPSw
appears in the cell lysate between 15 and 25 min
of chase. By 90 min, little iAPPSw
is still detectable.
B, secreted APPSw
is not detected in the conditioned
medium until after 35-45 min of chase. C, the appearance
and disappearance of iAPPSw
coincides with that observed
for the mature form of full-length APPSw in cell lysates.
ELISA Is a Specific and Sensitive Detector of
Inhibitors of
-Secretase Cleavage--
The serine protease
inhibitor AEBSF, inhibits A
-peptide secretion in a
concentration-dependent manner in both neuronal and non-neuronal cell lines stably expressing APP or APPSw by presumably blocking
-secretase cleavage (11). The APPSw
ELISA enabled us to
compare the concentration dependence of the AEBSF-induced inhibition of
-secretase activity to that reported for inhibition of A
peptide
secretion. CHOAPPSw cells plated the previous day were rinsed in PBS
and incubated for 5 h with fresh medium containing increasing
concentrations of AEBSF (0.1-1.0 mM). The conditioned medium was then collected, and APPSw
levels were quantified using the 931 ELISA. AEBSF produced a concentration-dependent
inhibition of APPSw
secreted into the medium (Fig.
5A). CHOAPPSw cells treated with 1 mM AEBSF had secreted only 43% of the APPSw
detected in conditioned medium from untreated cells. This supports the
observation that AEBSF inhibits
-secretase cleavage of APPSw by a
direct or indirect mechanism.
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Fig. 5.
The effects of protease inhibitors on
APPSw secretion in CHOAPPSw cells.
A, CHOAPPSw cells were incubated for 5 h in complete
DMEM containing either vehicle or 0.1-1.0 mM AEBSF, a
serine protease inhibitor. Conditioned medium was collected and
analyzed by ELISA for APPSw
secretion. B, CHOAPPSw cells
were incubated for 6 h with vehicle or 1-20 µM
clasto-lactacystin
-lactone, a specific inhibitor of the
proteasome. Secreted APPSw
levels were measured in triplicate by
ELISA.
and intracellular and secreted A
42 (18, 23, 24). Other
investigators reported that lactacystin increased secretion of both
A
40 and A
42 from SH-SY5Y cells (14). The proposed explanation for
these increases is that inhibition of the proteasome prevents
degradation of both full-length and COOH-terminal fragments of APP
making more of each available for cleavage by the
- and
-secretases, respectively (18). We reasoned that, if lactacystin disrupted ER-associated degradation of APPSw in a manner similar to
other transmembrane proteins (25-28), then more full-length protein
would also be available for cleavage by
-secretase. To test this
hypothesis, CHOAPPSw cells were incubated for 6 h with increasing
concentrations of the active form of lactacystin,
clasto-lactacystin
-lactone (
-lactone). This
cell-permeable, irreversible inhibitor of the proteasome has a 5-10
fold greater potency than lactacystin with an IC50 in
intact cells of 1 µM (29). ELISA measurements of secreted
APPSw
revealed that a concentration of 10 times the IC50
(i.e. 10 µM) still had no effect on the levels
of secreted APPSw
(Fig. 5B). Only at a concentration of
20 µM
-lactone was a decrease in
-secretase-cleaved
APPSw detectable (about 85% of control levels). Thus, proteasomal
inhibition has little influence on
-secretase cleavage of APPSw.
peptide secretion at low concentrations and inhibit A
secretion at
high concentrations (13-17). MG132 was one of the most potent peptide
aldehyde inhibitors of A
peptide secretion (15, 18). We hypothesized
that MG132 was inhibiting A
peptide secretion at high concentrations
by interfering with
-secretase cleavage of APP. To test this
hypothesis, CHOAPPSw cells were incubated for 6 h with MG132 at
concentrations demonstrated to inhibit A
peptide secretion (20-100
µM). The 931 ELISA was used to quantify the amount of
APPSw
secreted by MG132-treated cells and compared with untreated
controls. MG132 treatment produced a concentration-dependent
decrease in the levels of APPSw
detected in the conditioned media
(Fig. 6A). Cells incubated
with 100 µM MG132 secreted APPSw
at 35% the level
secreted by untreated cells. As a control, MG132 was added to a final
concentration of 100 µM following collection of media
that had been conditioned for 6 h by untreated cells (100+). The
level of APPSw
was identical to untreated samples, indicating that
MG132 was not interfering with the ELISA itself. To determine whether
MG132 was inhibiting APPSw
levels by decreasing the levels of
full-length APPSw, the 945 ELISA was performed on equal amounts of
lysates of the treated cells. No significant difference in full-length
APPSw levels was detected with MG132 treatment (Fig. 6B).
Thus, MG132 treatment caused a concentration-dependent
decrease in the amount of secreted APPSw
. To examine the time course
of this block in APPSw
production, CHOAPPSw cells were incubated for
increasing time with or without 80 µM MG132. Conditioned
medium was collected at the times indicated and the amount of secreted
APPSw
determined by ELISA (Fig. 6C). Our results show
that inhibition of APPSw
secretion could not be detected until
2 h following addition of the peptide aldehyde inhibitor.
Following this incubation period, APPSw
continued to be secreted
into the media by MG132-treated cells but at a much slower rate
compared with untreated controls.
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Fig. 6.
The peptide aldehyde, MG132, inhibits
-secretase cleavage of APPSw in stably transfected
CHO cells. A, CHOAPPSw cells were incubated for 6 h with Me2SO vehicle or 20-100 µM MG132.
Conditioned medium was collected and analyzed for secreted APPSw
by
ELISA. To control for any interference with the ELISA, MG132 was added
to a final concentration of 100 µM after CHOAPPSw cells
had conditioned medium for 6 h (white bar
labeled 100+). B, an ELISA was also conducted on
10-µg aliquots of CHOAPPSw cell lysates to determine whether MG132
treatment altered levels of APPSw in the cells. C, MG132
inhibits secretion of APPSw
in a time-dependent manner.
CHOAPPSw cells were incubated with either Me2SO vehicle
(solid line) or 80 µM MG132
(dashed line) for 0, 0.5, 1, 2, 4, 6, or 8 h. Conditioned medium was collected and the levels of secreted APPSw
determined by ELISA.
secretion 30 min and 1 h after
incubation with MG132 could be explained in two ways. Either this was
the time required for MG132 to penetrate the cell and block
-secretase cleavage or inhibition was immediate but was not
detectable by this assay because of the lag time required for secretion
of APPSw
generated prior to the block. It was also of interest to
determine whether the block in APPSw
production by MG132 was similar
in its immediacy and specificity to that observed for AEBSF. Therefore,
CHOAPPSw cells were pulsed for 12 min with Tran35S-label
and then chased for 0, 45, or 90 min in complete medium containing
Me2SO, 80 µM MG132, or 1 mM
AEBSF. The resulting cell lysates were immunoprecipitated first with
931 followed by 945. Equal aliquots of conditioned medium were
incubated with 931 or 6E10 to immunoprecipitate secreted APPSw
and
APPSw
, respectively. When 80 µM MG132 was present only
during the chase period, it had little effect on full-length APPSw
maturation, intracellular APPSw
production, or APPSw
secretion
compared with control. MG132 treatment, however, produced a large
increase in the amount of secreted APPSw
(Fig.
7D). In contrast to this is
the effect of chasing in the presence of 1 mM AEBSF, which
immediately blocked the appearance of APPSw
in the medium (Fig.
7C). The amount of iAPPSw
was markedly reduced
after chasing for 45 min compared to control and was still reduced
after 90 min (Fig. 7B). AEBSF treatment also stabilized the
immature form of full-length APPSw, which was nearly unchanged through
90 min of chase in the presence of the inhibitor (Fig. 7A).
Although AEBSF nearly completely blocked APPSw
secretion, secreted
levels of APPSw
were slightly increased after 90 min of chase (Fig.
7D). The lack of an effect of MG132 during this chase period
indicates that it does not produce an immediate block in cleavage as
observed with AEBSF. These results suggest that, although both AEBSF
and MG132 induced a block in APPSw
production, they differ in their
molecular mechanisms.
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Fig. 7.
A comparison of AEBSF and MG132 incubation on
intracellular production of APPSw .
CHOAPPSw cells were pulsed for 12 min with Tran35S-label.
The cells were washed and then chased in complete DMEM containing
Me2SO, 80 µM MG132, or 1 mM AEBSF
for either 45 or 90 min as described under "Experimental
Procedures." Intracellular APPSw
indicated by an arrow
(panel B) and full-length APPSw (panel
A) were isolated from cell lysates by sequential
immunoprecipitation with 931 and 945 antisera, respectively.
Conditioned medium was isolated and analyzed for secreted APPSw
(panel C) and APPSw
(panel
D) by immunoprecipitation using the 931 and 6E10 antibodies,
respectively.
in the first 90 min of
incubation (see Figs. 6 and 7). Therefore, an additional pulse-chase
experiment was conducted in which cells were preincubated for 2 h
with 80 µM MG132 or Me2SO since a block in
APPSw
production was detected after 2 h of MG132 treatment
(Fig. 6C). After the pretreatment, cells were pulsed and
then chased in the presence of vehicle or MG132. Conditioned media and
cell lysates were isolated and analyzed as above for full-length APPSw,
iAPPSw
, APPSw
, and APPSw
. A profound effect was
observed on APPSw processing after 2 h of MG132 preincubation
(Fig. 8). After a 45-min chase, mature
APPSw was readily detectable in CHOAPPSw cells treated with
Me2SO, while the MG132-treated cells showed only a trace of
the mature form (Fig. 8A). After 90 min of chase, very
little radiolabeled immature or mature APPSw remained in the control sample, whereas in the MG132-treated cells a large portion of the
radiolabeled immature APPSw detected at time zero still remained. Corresponding with this impaired maturation of APPSw with MG132 treatment was a decrease in the levels of iAPPSw
(Fig.
8B), secreted APPSw
(Fig. 8C), and secreted
APPSw
(Fig. 8D). This suggests that treatment of cells
with high concentrations of peptide aldehydes may cause a more general
impairment of the secretory pathway. This impairment blocks transport
and processing of APPSw through the secretory pathway, thus explaining
the decrease in A
peptide secretion observed by others (12-14).
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Fig. 8.
A 2-h preincubation with MG132 blocks
intracellular production of APPSw in CHOAPPSw
cells. CHOAPPSw cells were preincubated for 2 h with either
Me2SO control or 80 µM MG132. The cells were
subsequently starved of methionine and cysteine, pulsed for 12 min with
Tran35S-label and chased all in the presence of either
Me2SO or 80 µM MG132 for either 45 or 90 min
as described under "Experimental Procedures." Intracellular
APPSw
indicated by an arrow (panel
B) and full-length APPSw (panel A)
were isolated from cell lysates by sequential immunoprecipitation with
931 and 945 antisera, respectively. Conditioned medium was isolated and
analyzed for secreted APPSw
(panel C) and
APPSw
(panel D) by immunoprecipitation using
the 931 and 6E10 antibodies, respectively.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and p3 peptide secretion by blocking
-secretase cleavage of the COOH-terminal fragment of APP (12, 13, 17, 18). This
conclusion was based on the observation that CTFs were detectable in
cells incubated with peptide aldehydes. Our data suggest that peptide
aldehydes, such as MG132, have a more widespread effect on APP
maturation, processing and secretion. First and foremost,
-secretase
cleavage of APPSw was blocked at concentrations of MG132 reported to
impair A
peptide secretion (15, 18). This inhibition in
-secretase cleavage is sufficient to account for the reported
decrease in A
peptide secretion. Second, we demonstrated that, after
2 h of incubation with MG132, secretion of APPSw
was also
inhibited. Finally, in pulse-chase studies on CHOAPPSw cells
preincubated with MG132, fully mature APPSw was barely detectable in
the cell lysates. Instead, the immature, N-glycosylated form
that is located in the ER was stabilized, suggesting MG132 incubation
has multiple effects on APPSw processing. Like Skovronsky et
al. (18), we were unable to detect stabilization of mature APPSw
and inhibition of secreted APPSw
and APPSw
, when cells were only
incubated with MG132 during the chase period. However, our studies, in
which a 2-h preincubation with MG132 blocked production of
iAPPSw
, may better represent the effects of peptide
aldehydes on A
peptide secretion over the long incubation period
used by these and other investigators (13-18).
-secretase cleavage along
with results of others describing the stabilization of CTFs in cell
lysates by such peptide aldehydes can be best explained if treatment
with these compounds is somehow impeding progression of APPSw through
the secretory pathway. Haass et al. (30) have shown that
impairing maturation of APPSw by treating cells with BFA prevents
production of intracellular APPSw
because transport to the site of
-secretase cleavage is blocked. We obtained the same results using
the 931 antisera described here. Other investigators achieved similar
results by blocking movement of APP from the endoplasmic reticulum
(ER). In those studies, a dilysine ER retrieval motif introduced into
the cytoplasmic tail of APP strongly impaired cleavage by
-secretase
(31). We, again, have obtained equivalent results expressing APPSw
bearing a dilysine ER retrieval motif in CHO
cells.2 Like BFA treatment or
introduction of a dilysine retrieval motif, a 2-h MG132 treatment of
CHO cells stably expressing APPSw blocked the maturation of the
full-length molecule in experiments described here. This, in turn,
blocked production of intracellular APPSw
and its subsequent
appearance in the media. We suggest that the increase in the C83 and
C99 CTFs observed by others with MG132 treatment results from a similar
block in access of these
- and
-secretase-cleaved fragments to
-secretase. Regardless of the mechanism, our results showing that
MG132 blocks
-secretase cleavage of APPSw call into question the
utility of such peptide aldehydes as
-secretase inhibitors.
and did not cross-react with full-length APPSw,
APPSw
, APP
, or APP
. This enabled us to detect intracellular APPSw
in stably transfected CHOAPPSw cells. The results observed in
our pulse-chase studies agree closely with previous findings of APPSw
processing in HEK293 cells (22). Intracellular APPSw
was detectable
within 25 min in pulse-chase experiments and coincided with the
appearance of mature APPSw. Furthermore, we clearly detected APPSw
intracellularly prior to its appearance in the medium, eliminating the
possibility that the APPSw
detected in cell lysates was due to
already secreted
-cleaved fragments associating with the cell membrane.
peptide by
inhibiting
-secretase activity to about 44% of untreated cells (11). Our results measuring the secretion of APPSw
using the 931 ELISA showed that 1 mM AEBSF reduced APPSw
secretion to
a remarkably similar 43% of control levels. Our results also agreed with theirs in that AEBSF treatment caused a slight increase in APPSw
secretion and stabilized full-length APPSw. Furthermore, we
extended these observations by showing that this inhibition of
-secretase cleavage had a concentration dependence similar to that
observed for A
peptide secretion (11). Only a small amount of
iAPPSw
was detected in AEBSF-treated cells. This showed that AEBSF inhibition was not due to accumulation of APPSw
intracellularly because of a block in secretion, but due to a block in
-secretase cleavage of APPSw. Knowing now that BACE is an aspartic
protease, it is unclear how a serine protease inhibitor such as AEBSF
may block
-secretase cleavage, but it would argue against a direct inhibition of BACE. However, the discovery of such an inhibitor illustrates the importance of an in vivo screening method
for compounds that block
- and
-secretase cleavage of APP.
peptide secretion have been complex and conflicting. Nevertheless, results have repeatedly shown that A
and p3 peptide secretion is
inhibited by the following peptide aldehydes: MDL23170 (12, 32), ALLN
(13, 15, 17), calpeptin (14, 15), and MG132 (15, 18) with the latter
proving to be one of the most potent. Because increased levels of CTFs
were detected in cells incubated with peptide aldehydes, the impairment
in subsequent A
peptide secretion has been attributed to an
inhibition of
-secretase activity by peptide aldehydes (12). Our
results using both an APPSw
ELISA and pulse-chase studies revealed
that
-secretase cleavage of APPSw was also impeded by MG132. The
concentration dependence of this
-secretase cleavage inhibition
closely corresponds to that reported for the decrease in A
40,
A
42, and p3 peptide secretion (15, 18). Inhibition of
-secretase
cleavage was detectable after as little as 2 h of exposure to
MG132. In studies by others (13-18), A
peptide levels were measured
after 3-16 h of incubation with peptide aldehyde. Thus, the
MG132-induced block in
-secretase cleavage that we observed here
could account for the decrease in A
peptide secretion. This
observation indicates that the inhibitory effect by peptide aldehydes
such as MG132 is not limited to inhibition of
-secretase activity.
In addition to inhibiting cysteine proteases, MG132 is known to inhibit
proteasomal cleavage with an IC50 of a few micromolar (29).
However, the potent and highly specific proteasomal inhibitor,
clasto-lactacystin
-lactone, only caused a partial
inhibition in APPSw
secretion in our studies at 20 times its
IC50 for proteasomal inhibition. Therefore, we conclude
that the decrease in
-secretase cleavage of APPSw is not due to
MG132 inhibition of proteasomal activity. In fact, lactacystin and low
concentrations of peptide aldehydes are reported to increase A
peptide secretion (14). We detected no increase in
-secretase
cleavage of APPSw with clasto-lactacystin
-lactone or low
concentrations of MG132 (data not shown). Thus, we suggest that low
concentrations of peptide aldehydes increase A
peptide secretion
because they inhibit proteasomal degradation of
and
-secretase-cleaved CTFs. This, in turn, makes more CTFs available
for
-secretase cleavage. We further hypothesize that, at higher
concentrations, peptide aldehydes inhibit A
and p3 peptide secretion
by blocking cysteine proteases that have roles in protein processing
and trafficking in the secretory pathway. This would explain the
biphasic effect of peptide aldehydes observed on A
peptide secretion
(14, 15, 17).
antibody, 931, may
prove even more useful in analyzing APPSw processing in neuronal cell
lines where greater levels of iAPPSw
may be expected. In
particular, using affinity-purified 931 in the APPSw
sandwich ELISA
described here will be immensely useful for exploring the cellular and
molecular mechanisms that regulate
-secretase cleavage.
![]() |
ACKNOWLEDGEMENTS |
---|
We gratefully thank Dr. Dale Schenk for the generous gift of the mouse monoclonal antibody, 8E5. We also thank Taraneh Haske for providing the two CHO cell lines stably expressing APPSw and APP wild type.
![]() |
FOOTNOTES |
---|
* This work was supported in part by a Michigan Alzheimer's Disease Research Center pilot grant (to J. R. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Recipient of an Institute of Gerontology training fellowship (through Grant T32AG00114 from NIA, National Institutes of Health).
To whom correspondence should be addressed: Inst. of
Gerontology and Dept. of Biological Chemistry, University of Michigan, 300 N. Ingalls, Rm. 973, Ann Arbor, MI 48109. Tel.: 734-764-3250; E-mail: jrgaut@umich.edu.
Published, JBC Papers in Press, November 17, 2000, DOI 10.1074/jbc.M008793200
2 M. L. Steinhilb, R. S. Turner, and J. R. Gaut, manuscript in preparation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
A peptide, a 40- or 42-amino acid peptide derived from APP;
p3, peptide derived from
- and
-secretase cleavage of APP;
AEBSF, 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride;
CTF, COOH-terminal fragment;
APP, amyloid precursor protein;
APPSw, APP
bearing the Swedish mutation (K651N/M652L);
BACE,
-site APP-cleaving
enzyme;
ELISA, enzyme-linked immunosorbent assay;
APPSw
, soluble
-secretase-cleaved APPSw fragment;
iAPPSw
, intracellular soluble
-secretase-cleaved APPSw fragment;
APPSw
, soluble
-secretase-cleaved APPSw fragment;
APP
, soluble
-secretase-cleaved APP fragment;
APP
, soluble
-secretase-cleaved APP fragment;
CHO, Chinese hamster ovary;
HEK293, human embryonic kidney;
PAGE, polyacrylamide gel electrophoresis;
DMEM, Dulbecco's modified Eagle's medium;
PBS, phosphate-buffered saline;
PBS/T, phosphate-buffered saline plus Tween 20;
ER, endoplasmic
reticulum;
BFA, brefeldin A.
![]() |
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