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INTRODUCTION |
The amyloidoses are complex, multiform disorders characterized by
the polymerization and aggregation of normally innocuous and soluble
proteins or peptides into extracellular insoluble fibrils. More than 16 biochemically unique proteins, including transthyretin,
-synuclein,
calcitonin,
2-macroglobulin, gelsolin, amylin, and
-amyloid, have been isolated as the fibrillar components of
disease-associated amyloid deposits (1-5). These proteins share no
conserved primary structural motives or other structural homologies,
but their fibrils all possess some common structural features (3, 6,
7). All amyloid fibrils contain
-sheet structures in which the
polypeptide chains are orthogonally aligned in the fibril directions
(8-10). With Congo Red staining, amyloids show a green birefringence
under polarized light, and under electron microscopy, their morphology
consists of bundles of nonbranching, long filaments about 5-12 nm wide
(4, 5, 11).
The most characterized amyloid-forming peptide is
-amyloid
(A
)1 of Alzheimer's
disease. The toxicity of A
has been directly linked to structure and
amyloid content. In an aggregated state (containing fibrils,
protofibrils, and low molecular weight intermediates), A
has been
consistently shown to be toxic to neurons in culture (12-18). Although
there is some disagreement as to the exact structure of the aggregated
species associated with toxicity, whether it be a protofibril (14, 19),
a diffusible, nonfibrillar ligand (20), or some other low molecular
weight intermediate (19), toxicity is associated with peptide
structures that are part of the aggregation pathway associated with
amyloid formation. In addition, A
neurotoxicity has been shown to be
attenuated by Congo Red and rifampicin, which bind to and selectively
inhibit the formation of A
amyloid fibrils (21-24). Clearly, all of
these observations imply a causal link between A
fibril formation
and neurodegeneration.
Various research groups have hypothesized potential molecular
mechanisms of
-amyloid toxicity, but there is no consensus. Cellular
responses to A
that have been postulated to result in toxicity
encompass destabilization of calcium homeostasis, membrane depolarization, increased vulnerability to excitotoxins, increased membrane permeability due to free radical generation, blockage or
functional loss of potassium channels, and direct disruption of
membrane integrity (17, 18, 25-36). The preceding plethora of observed
biochemical responses to A
suggests that perhaps a more common,
fundamental pathway is initially being activated and that this pathway
subsequently diverges to produce many unique intracellular responses.
Analogous to A
, calcitonin is another model amyloid peptide
associated with medullary carcinoma of the thyroid (37-43). The fibrils of human calcitonin have also been shown to be neurotoxic (44-46), suggesting that the amyloidoses may possess a shared
mechanism of toxicity related to their secondary and macromolecular structures.
To explore if a common, fundamental mechanism of toxicity exists in the
amyloidoses, we examined the structure-function relationships of
several synthetic A
sequences, A
-(1-40), A
-(25-35),
A
-(1-16), and bovine calcitonin. We were able to manipulate the
secondary and macromolecular structures of these peptides to produce
stable amyloid and nonamyloid structures. With these model systems, we demonstrated that the peptides in an amyloid state (with high
-sheet
content and the ability to bind Congo Red) altered G protein activity
associated with both cell membrane extracts and purified G
subunits.
We showed that the abilities of the peptides to induce GTPase
activation were correlated with their toxicities, and the neurotoxicities of the peptides were attenuated by specific and nonspecific GTPase inhibitors. In addition, we demonstrated that significant GTPase activities were still induced even when the cell
surface receptors were removed with a nonspecific protease. These
results suggest that G protein activation, possibly induced via a
protein-membrane interaction, plays an important role in the toxicity
of A
and other amyloid-forming proteins.
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EXPERIMENTAL PROCEDURES |
Materials--
A
-(1-40), A
-(25-35), and A
-(1-16)
were purchased from BIOSOURCE International
(Camarillo, CA), and bovine calcitonin was obtained from Sigma. ATP and
GTP were purchased from Aldrich, and [
-32P]GTP was
from ICN Biochemicals (Irvine, CA). Suramin and Pronase were acquired
from Calbiochem and Roche Molecular Biochemicals, respectively. Cell
culture reagents were purchased from Life Technologies, Inc. Purified
1,2-dipalmytoyl-sn-glycero-3-phosphocholine (DPPC) and
cholesterol were obtained from Avanti Polar Lipids (Alabaster, AL). The
XK16/70 column and Superfine Sephadex G-50 for size exclusion were
acquired from Amersham Pharmacia Biotech. Purified Go and Gi
subunits and epinephrine were purchased from
Calbiochem. All other chemicals, unless otherwise specified, were
obtained from Sigma.
A
Peptide Preparation--
The A
peptides were prepared
analogously to established methods in the toxicity and structural
literature for forming
-sheet structures and fibril formations
(47-50). Stock solutions of 10 mg/ml were prepared by dissolving the
A
peptides in 0.1% (v/v) trifluoroacetic acid in water. After
incubating for 1 h at 25 °C, the peptide stock solutions were
diluted to concentrations of 0.5 mg/ml in sterile phosphate-buffered
saline (PBS) (0.01 M NaH2PO4, 0.15 M NaCl, pH 7.4) with antibiotics. These solutions were
rotated on a model RD4524 rotator (Glas-col, Terre Haute, IN) at 60 rpm
at 25 °C for 24 h. The peptides were then diluted to final
concentrations of 20 µM in sterile medium and rotated for
an additional 24 h prior to being added to the culture wells or
plates for the toxicity and GTP studies.
The Structural Characterization and Preparation of Bovine
Calcitonin--
Bovine calcitonin was directly dissolved in various
solvents and buffers at concentrations of 40 and 80 µM.
CD measurements of the solutions were recorded 2-24 h later on a model
62DS spectrometer (Aviv Instruments, Lakewood, NJ) at 25 °C using a
bandwidth of 1.0 nm, a step interval of 0.5 nm, and an averaging time
of 2 s. A 0.01-cm quartz cell was used for the far-UV (190-250
nm) measurements. The instrument was calibrated using
D(+)-10-camphorsulfonic acid. Three scans each of duplicate
samples were measured and averaged. Control buffer and solvent scans
were run in duplicate, averaged, and then subtracted from the sample
spectra. Spectra were analyzed using the secondary structural
parameters reported by Chang (51) to ascertain the sample percentages
of
-helix,
-sheet,
-turn, and random-coil.
To assess the presence of amyloid fibrils in the calcitonin solutions,
Congo Red binding studies were performed. Congo Red dye was dissolved
in PBS to a final concentration of 112 µM. Congo Red
absorbances of the calcitonin solutions and free dye controls were
determined by adding Congo Red to a final concentration of 12 µM and acquiring spectral measurements from 300 to 900 nm
at 25 °C on a model 420 UV-visible spectrophotometer (Spectral
Instruments, Tucson, AZ) (52, 53). Both the calcitonin solutions and
the control solutions were allowed to interact with Congo Red for 1 h prior to recording their spectra. Congo Red difference spectra were calculated by subtracting the free dye absorbance from the calcitonin-dye absorbances.
Finally, bovine calcitonin was prepared in the following manner for the
toxicity and GTP studies. Calcitonin was dissolved at a concentration
of 1.5 mg/ml in either deionized water or a solution of 5 mM CaCl2 and 1 mM MgCl2
in water. These solutions were rotated at 60 rpm at 25 °C for
24 h. Then the calcitonin solutions were diluted to 40 and 80 µM with sterile medium and rotated for an additional
24 h prior to being added to the culture wells or plates for the
toxicity and GTP studies.
Cell Culture--
Human neuroblastoma SH-SY5Y cells (a gift of
Dr. Evelyn Tiffany-Castiglioni, College of Veterinary Medicine, Texas A
& M University, College Station, TX) were cultured in a humidified 5%
(v/v) CO2/air environment at 37 °C in minimum Eagle's
medium supplemented with 10% (v/v) fetal bovine serum, 3 mM L-glutamine, 100 units/ml penicillin, 100 µg/ml streptomycin, and 2.5 µg/ml amphotericin B (fungizone). Likewise, rat pheochromocytoma PC12 cells (ATCC, Manassas, VA) were
cultured in RPMI medium supplemented with 10% (v/v) horse serum, 5%
(v/v) fetal bovine serum, 3 mM L-glutamine, 100 units/ml penicillin, 100 µg/ml streptomycin, and 2.5 µg/ml
fungizone in a 5% (v/v) CO2/air environment at 37 °C.
For the GTP studies, cells were plated at densities ranging from 2.5 to
4 million cells in 35-mm tissue culture dishes. For the viability
assays, cells were plated at a density of 1 × 105
cells/well in 96-well plates. During both the GTP and viability studies, the peptides were added to the cells 24 h after plating.
Membrane Preparation for the GTPase Assay--
After incubation
with the peptides or controls for 30 min at 37 °C, the PC12 or
SH-SY5Y membranes were isolated using the widely accepted method of
Seifert and Schultz (54). Cells were harvested with a cell scraper and
collected by centrifugation (1600 × g, 4 °C, 20 min). Subsequently, they were washed with a buffer consisting of 10 mM triethanolamine (TEA) and 140 mM NaCl (pH
7.4) and disrupted by nitrogen cavitation in a 50 mM KH2PO4 buffer with 100 mM NaCl, 3 mM EDTA, and 15 mM
-mercaptoethanol (pH
7.0). The nuclear portion of the cells was removed by a short centrifugation (1000 × g, 4 °C, 2 min), and
membrane sedimentation was attained with a long centrifugation
(15,000 × g, 4 °C, 60 min). The resulting membrane
pellet was suspended in a 10 mM TEA/HCl buffer (pH 7.4),
and the total membrane protein content was measured with the BCA assay
(55).
GTPase Assay--
The GTPase activities of the PC12 and SH-SY5Y
membranes were assessed similarly to procedures described previously
(56-60). Reaction mixtures of 100 µl consisted of 0.4 µM [
-32P]GTP (0.5 µCi/tube), 0.5 mM MgCl2, 0.1 mM EGTA, 0.1 mM ATP, 1 mM AMP-PNP, 5 mM creatine
phosphate, 40 µg of creatine kinase, 1 mM dithiothreitol,
and 0.2% (w/v) bovine serum albumin in 50 mM TEA/HCl (pH
7.4). Following a 5-min preincubation period at 25 °C, the reaction
was initiated by the addition of 5-8 µg of membrane protein. After
15 min at 25 °C, the reaction was stopped by the addition of 800 µl of a 20 mM KH2PO4 buffer
(4 °C, pH 7.0) containing 5% (w/v) activated charcoal. The released
32Pi was separated from the nucleotide-bound
phosphate by centrifugation (15000 × g, 4 °C, 20 min), and 100 µl of the supernatant was counted on a Topcount
Microplate Scintillation Counter (Packard Instrument Co.).
Low affinity or nonspecific GTPase activity was measured by adding
excess unlabeled GTP (50 µM) to the aforementioned
reaction mixture and conducting the reaction as described. Specific
high affinity GTPase activity was calculated as the difference between the total GTPase activity in the absence of unlabeled GTP and the low
affinity GTPase activity.
Pronase Experiments--
Pronase studies were conducted
analogously to established procedures (61-63). Pronase at a
concentration of 3 mg/ml in serum-free RPMI was incubated with the
plated PC12 cells for 1 h at 4 °C. The cells were harvested
with a cell scraper and collected by centrifugation (1600 × g, 4 °C, 20 min). Then the cells were thoroughly washed
with PBS and centrifuged again (1600 × g, 4 °C, 20 min) prior to the addition of the peptides or controls for the GTPase assays. For the epinephrine control, 200 µM epinephrine
in serum-free RPMI was incubated with these Pronase-treated PC12 cells
for 30 min prior to the membrane isolation step.
MTT Reduction Assay--
SH-SY5Y and PC12 cell viability was
measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) reduction assay. MTT is reduced by viable cells to form
blue formazan crystals, and inhibition of this reaction is indicative
of cellular redox alterations that could result in toxicity (64). The
peptides were incubated with the SH-SY5Y and PC12 cells for 24 h,
after which time MTT reduction was assessed. MTT was added to the
culture medium to yield a final concentration of 0.5 mg/ml. The cells were allowed to incubate with the MTT for 4 h in a CO2
incubator after which time 100 µl of a 5:2:3
N,N-dimethylformamide/SDS/water solution (pH 4.7) was added
to dissolve the formed formazan crystals. After 18 h of incubation
in a humidified CO2 incubator, the results were read using
an Emax Microplate reader at 585 nm (Molecular Devices, Sunnyvale, CA).
Viability is reported relative to control cells unexposed to the peptides.
GTPase and Toxicity Inhibition--
Pertussis toxin (PT) (100 ng/ml), GDP
S (600 µM), and suramin (20 µM) were incubated with the PC12 and SH-SY5Y cells for
24, 3, and 3 h, respectively, at 37 °C prior to the peptide
additions for the GTPase or toxicity assays. The peptide solutions for
these assays also contained the same inhibitors at the same
concentrations. Control cells were treated identically except for the
presence of peptide.
Reconstitution of G
o and G
i
Vesicles--
Vesicles consisted of 82% (w/w) DPPC and 18% (w/w)
cholesterol, and they were prepared by mixing the DPPC and cholesterol in chloroform and evaporating off the solvent under nitrogen at 50 °C in a 421-4000 Micro Rotary Evaporator (Labconco, Kansas City,
MO). 20 mM NaHepes (pH 8.0) containing 0.4% (w/v)
deoxycholate and 0.04% (w/v) cholate was then added to suspend the
lipid film, producing a final lipid concentration of 1 mg/ml. The
resulting DPPC/cholesterol suspension was sonicated for 10 min.
Subsequently, 1.2 volumes of these DPPC/cholesterol vesicles were
combined with 0.6 volume of G
o or G
i in a
10 mM NaHepes buffer (pH 8.0) containing 1 mM
EDTA, 0.1 mM dithiothreitol, and 0.1% (v/v) Genapol. This mixture was gel-filtered using an ÄKTA Explorer (Amersham
Pharmacia Biotech) with Sephadex G-50 in a XK16/70 column at a flow
rate of 0.5 ml/min according to procedure of Pedersen and Ross (65). The elution buffer consisted of 20 mM NaHepes buffer (pH
8.0), 1 mM EDTA, 1 mM dithiothreitol, 0.1 M NaCl, and 2 mM MgCl2.
G
GTPase Assay--
GTPase activity was assayed by incubating
the G
o or G
i vesicles (5 µl; 75 fmol of
G
o or G
i) at 30 °C in a total volume of 100 µl containing 50 mM NaHepes (pH 8.0), 1 mM EDTA, 1 mM dithiothreitol, 0.1 M
NaCl, 2 mM MgCl2, 0.1 mM adenosine
AMP-PNP, 0.1 mM ascorbic acid, and 0.1 µM
[
-32P]GTP. After the specified time, the reaction was
stopped by the addition of 250 µl of a 50 mM
NaH2PO4 buffer (4 °C, pH 7.0) with 5% (w/v)
activated charcoal and rapid chilling. The mixture was centrifuged
(1200 × g, 4 °C, 10 min), and the
[32P]Pi in the supernatant (200 µl) was
counted on a Topcount Microplate Scintillation Counter (Packard
Instrument Co.).
Statistical Analysis--
The significance of results was
determined using a Student's t test on n
independent measurements, where n is specified in the figure
legend. Unless otherwise indicated, significance was taken as
p < 0.05.
 |
RESULTS |
Peptide Secondary and Macromolecular Structures--
The
structures of the A
peptides under the employed solvation conditions
have been well characterized and were not re-examined. Under these
conditions, A
-(1-40) and A
-(25-35) have been shown to be
amyloidogenic (containing fibrils and protofibrils) and to contain
extensive
-sheet structures, whereas A
-(1-16) has been
demonstrated to be nonamyloidogenic and predominantly random coil
(47-50).
Because the solution structures of bovine calcitonin are not as well
documented, we identified conditions that promoted the formation of
-sheet structure and amyloid using CD spectroscopy and Congo Red
binding assays. As determined by CD (Fig.
1A), both 40 and 80 µM bovine calcitonin in water containing 5 mM
CaCl2 and 1 mM MgCl2 adopted
structures with ~55 ± 10%
-sheet and only 15 ± 10%
-helix. Incubating the peptide in deionized water alone at these
same concentrations (Fig. 1A) produced structures devoid of
-sheet character with 95 ± 10%
-helical contents. As
depicted by Congo Red difference spectra (Fig. 1B), the 40 and 80 µM water solutions of bovine calcitonin with 5 mM CaCl2 and 1 mM MgCl2 significantly bound and shifted the spectral properties of Congo Red,
indicating the formation of amyloid. The zero difference spectrum of
the peptide in deionized water indicated an absence of Congo Red
binding and substantial amyloid fibril formation (spectrum not
shown).

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Fig. 1.
The structural characterization of bovine
calcitonin. A, representative CD spectra illustrating
that 80 µM calcitonin dissolved in deionized water ( )
was predominantly -helical, whereas both 40 ( ) and 80 µM ( ) calcitonin dissolved in water containing 5 mM CaCl2 and 1 mM MgCl2
were enriched in -sheet character. B, absorbance
difference spectra for Congo Red indicated that 40 ( ) and 80 µM ( ) calcitonin dissolved in water with divalent
cations were amyloidogenic.
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|
GTPase Activity--
By using A
-(1-40), A
-(25-35), and
bovine calcitonin in water with 5 mM CaCl2 and
1 mM MgCl2 as models of peptides with
amyloidogenic structures and A
-(1-16) and bovine calcitonin in
deionized water as models of peptides without amyloidogenic structures,
we examined the relationship between peptide structure and GTPase
activity. We found that the rate of high affinity GTP hydrolysis in
PC12 membranes increased by 31 ± 12% on average with exposure to
the peptides containing extensive
-sheet and amyloid contents
relative to the rate of hydrolysis of control cells unexposed to
peptides (Fig. 2A). In all
cases, the increases in GTPase activity were significant relative to
the control cells (p < 0.001). The rates of GTP
hydrolysis were significantly greater for A
-(25-35) and 80 µM calcitonin (p < 0.001), the two
peptides with the greatest amyloid content, than for A
-(1-40) and
40 µM calcitonin, suggesting that the extent of
macromolecular structure influences the process. Similarly,
A
-(1-16) and bovine calcitonin in deionized water, the peptides
devoid of amyloid and
-sheet structures, did not significantly alter
the GTPase activities of the PC12 cells relative to untreated controls
(Fig. 2A) (p > 0.2).

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Fig. 2.
The GTPase activities of the
A and calcitonin peptides as a function of
their solution structures. PC12 (A) and SH-SY5Y
(B) cells were exposed to the peptides or controls for 30 min at 37 °C in a humidified 5% CO2 environment prior
to the GTPase membrane isolation step. The means ± S.D. of 3-8
determinations are depicted. Only the peptides with significant
-sheet and amyloid contents increased the high affinity GTP
hydrolysis. *, **, and *** indicate that the increases in the rates of
hydrolysis relative to the untreated control cells were significant at
p < 0.002, p < 0.0005, and
p < 0.0001, respectively.
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|
To ensure that the observed phenomena were not isolated to PC12 cells,
we examined the GTPase activities of SH-SY5Y membranes exposed to
bovine calcitonin. We found similar trends to those observed with the
PC12 cells (Fig. 2B). The rate of GTP hydrolysis increased
from 16.0 ± 0.5 pmol/mg/min for the control cells to 23.8 ± 0.6 and 18.7 ± 0.6 pmol/mg/min for the cells exposed to 80 and 40 µM bovine calcitonin in water with 5 mM
CaCl2 and 1 mM MgCl2, respectively
(p < 0.002). The nonamyloidogenic 80 µM calcitonin in water did not significantly alter GTPase activity relative to the controls (16.0 ± 0.3 pmol/mg/min,
p > 0.4).
Toxicity--
In parallel to the GTPase activity experiments, we
examined the relationship between peptide structure and toxicity using our model peptides. As seen in Fig. 3,
A and B, analogous to our GTPase results, we
found that exposure to the peptides containing extensive
-sheet and
amyloid contents resulted in significant PC12 (Fig. 3A) and
SH-SY5Y (Fig. 3B) cell toxicity (p < 0.001). Conversely, A
-(1-16) and bovine calcitonin in deionized
water, the peptides devoid of amyloid and
-sheet structures, did not significantly alter cell viability relative to untreated controls (p > 0.2).

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Fig. 3.
The toxicities of the A
and calcitonin peptides as a function of their solution
structures. The abilities of the PC12 (A) and SH-SY5Y
(B) cells to reduce MTT were taken as indications of cell
viability. The data are reported as the percentage of the MTT reduced
by the cells treated with the A and calcitonin solutions for 24 h relative to the MTT reduced by the cells untreated with these
solutions. The means ± the S.D. of 8-10 determinations are
depicted. The A -(1-40), A -(25-35), and calcitonin/salt
solutions, which contained substantial amyloid contents and -sheet
structures, always significantly inhibited the ability of the cells to
reduce MTT (*, p < 0.001), but the nonamyloidogenic
bovine calcitonin in deionized water and A (1-16) did not
significantly alter cell viability (p > 0.2).
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GTPase Inhibition--
To identify the family or families of G
proteins activated by the amyloid-forming peptides, we investigated the
effects of GDP
S and suramin, two nonspecific G protein inhibitors
(66-73), and PT, a specific inhibitor of the Gi and
Go families of G proteins (74-78). As illustrated in Fig.
4A, GDP
S, suramin, and PT
were each able to inhibit significantly the increases in GTP hydrolysis observed in PC12 membranes exposed to the amyloidogenic A
-(1-40), A
-(25-35), and bovine calcitonin (p < 0.005).
Analogously, the inhibitors significantly reduced the rate of GTP
hydrolysis induced by amyloidogenic calcitonin in membranes from
SH-SY5Y cells (Fig. 4B, p < 0.001).

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Fig. 4.
The effect of pharmacological agents on
GTPase activity. G protein inhibitors consistently reduced the
high affinity GTP hydrolysis of the PC12 (A) and SH-SY5Y
(B) cells caused by the amyloidogenic A peptides and
bovine calcitonin. The means ± S.D. of 3-6 determinations are
depicted. Untreated PC12 or SH-SY5Y cells were exposed to the amyloids
for 30 min at 37 °C in a humidified 5% CO2 environment
in the absence of all pharmacological agents. Treated PC12 or SH-SY5Y
cells were preincubated with 100 ng/ml PT, 20 µM suramin,
or 600 µM GDP S for 24, 3, and 3 h, respectively,
at 37 °C in a humidified 5% CO2 environment. Following
this preincubation, treated cells were exposed to amyloidogenic peptide
solutions containing the same inhibitors for 30 min at 37 °C in a
humidified 5% CO2 environment prior to the GTPase membrane
isolation step. PT (open bars), suramin (diagonally
striped bars), and GDP S (cross-hatched bars)
significantly reduced membrane GTPase activity relative to the cells
exposed to the amyloids in the absence of these compounds (solid
bars). * and ** indicate that the decreases in the rates of
hydrolysis relative to the untreated cells were significant at
p < 0.005 and p < 0.001, respectively.
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Pronase Experiments--
To ascertain if the peptide-induced
increases in GTPase activity were mediated through something other than
a peptide-receptor interaction, GTPase studies were performed with PC12
cells treated with Pronase. Pronase is a nonspecific protease that has
been documented to remove cell surface receptors (61-63). As shown in Fig. 5, GTP hydrolysis still increased
significantly in the presence of the amyloidogenic bovine calcitonin,
A
-(1-40), and A
-(25-35) relative to control cells treated with
Pronase but unexposed to the peptides (p < 0.003). The
rates of GTP hydrolysis for the Pronase-treated cells incubated with
A
-(1-40), A
-(25-35), and 80 µM bovine calcitonin
in water with divalent cations were increased by 9, 17, and 25%
respectively, compared with the Pronase-treated control cells. We also
examined the GTP hydrolysis of Pronase-treated cells with the
nonamyloidogenic A
-(1-16) and bovine calcitonin in deionized water
(Fig. 5). As expected, these peptides did not significantly alter
GTPase activity relative to the control cells (p > 0.15).

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Fig. 5.
The effect of Pronase-treatment on GTPase
activity. PC12 cells were incubated with 3 mg/ml Pronase in
serum-free RPMI media for 1 h at 4 °C. After washing and
centrifugation, the Pronase-treated cells were replated with the
peptide solutions or controls and incubated for 30 min at 37 °C in a
humidified 5% CO2 environment prior to the GTPase membrane
isolation step. The means ± the S.D. of 3-5 determinations are
depicted. Only the amyloidogenic peptides significantly increased GTP
hydrolysis relative to the Pronase-treated control cells. The absence
of increased GTP hydrolysis in the presence of epinephrine indicated
that the Pronase treatment effectively removed the cell surface
receptors. * and ** indicate that the increases in the GTPase activity
relative to the Pronase-treated control cells were significant at
p < 0.003 and p < 0.0001, respectively.
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To demonstrate the effectiveness of Pronase treatment at receptor
removal, we incubated cells with 200 µM epinephrine and then measured the rate of GTP hydrolysis. Without Pronase treatment, 200 µM epinephrine increased the rate of GTP hydrolysis
in PC12 cells by 100% (data not shown). However, as seen in Fig. 5,
after Pronase removal of cell receptors, incubation with epinephrine did not significantly alter the rate of GTP hydrolysis in the Pronase-treated cells relative to Pronase-treated control cells (p > 0.3). These results indicate that the cell
receptors had been effectively removed by the Pronase.
G
o and G
i GTPase Assays--
To
demonstrate more specifically that the peptides were interacting with
heterotrimeric G proteins, we performed GTPase assays with lipid
vesicles containing purified G
o and G
i
subunits (Fig. 6, A and
B). Analogous to the cell membrane GTP results, the
amyloidogenic A
-(1-40), A
-(25-35), and 80 µM
bovine calcitonin significantly increased GTPase activity in both the
G
o and G
i vesicles (p < 0.001). In contrast, A
-(1-16) and bovine calcitonin in deionized water, the peptides devoid of amyloid and
-sheet structures, did not
significantly stimulate GTPase activity in either vesicle system at any
of the experimental times. No receptors were included in the G
preparations.

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Fig. 6.
The GTPase activities of the
G o vesicles (A)
and the G i vesicles
(B). The production of
32Pi as a function of time, an indication of
GTPase activity, is shown. Vesicles (5 µl; 75 fmol of
G o or G i) were incubated at 30 °C
for the times shown. The amyloidogenic 20 µM A -(1-40)
( ), 20 µM A -(25-35) ( ), and 80 µM
bovine calcitonin in water containing 5 mM
CaCl2 and 1 mM MgCl2 ( )
stimulated GTPase activity in both the G o and
G i vesicles, but the nonamyloidogenic 20 µM A -(1-16) ( ) and 80 µM bovine
calcitonin in deionized water ( ) did not significantly affect GTPase
activity in either vesicle system at any of the experimental times.
Hydrolysis in the absence of vesicles is also shown ( ). The data
presented are averages of triplicate determinations that varied by less
than 10%, and the standard deviations of the determinations are
indicated by error bars where significant. The zero time,
zero protein values represent 32Pi
contaminating the [ -32P]GTP.
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Toxicity Inhibition--
To explore if the observed GTPase
activity was potentially linked to neurotoxicity, we examined if the
GTPase inhibitors attenuated the cell death caused by amyloidogenic
bovine calcitonin, A
-(1-40), and A
-(25-35). GDP
S, suramin,
and PT each significantly attenuated amyloid-induced PC12 cell toxicity
as seen in Fig. 7A
(p < 0.0005). Untreated cells exposed to A
-(1-40),
A
-(25-35), and 80 µM bovine calcitonin in water with
divalent cations were 52 ± 7% viable on average, but the cells
treated with GDP
S, suramin, and PT had viabilities of 83 ± 3, 81 ± 3, and 82 ± 4%, respectively. Comparable
neuroprotective trends were observed in SH-SY5Y cells (Fig.
7B).

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Fig. 7.
The influence of pharmacological agents on
cell viability as measured by MTT reduction. The inhibition of G
protein activation with PT, GDP S, and suramin attenuated the cell
death caused by the amyloidogenic bovine calcitonin, A -(25-35), and
A -(1-40) in PC12 (A) and SH-SY5Y cells (B).
Untreated PC12 and SH-SY5Y cells were exposed to the peptides for
24 h at 37 °C in a humidified 5% CO2 environment
in the absence of all pharmacological agents. Treated PC12 or SH-SY5Y
cells were preincubated with 100 ng/ml PT, 20 µM suramin,
or 600 µM GDP S for 24, 3, and 3 h, respectively,
at 37 °C in a humidified 5% CO2 environment. Following
this preincubation, treated cells were then exposed to amyloidogenic
peptide solutions containing the same inhibitors for 24 h at
37 °C in a humidified 5% CO2 environment cells. The
data are reported as the percentage of the MTT reduced by the cells
incubated with the peptides alone or with the peptides and inhibitors
relative to the MTT reduced by control cells unexposed to the peptides.
The means ± S.D. of 8 determinations are presented. In the
absence of pharmacological reagents, exposure to the amyloid-forming
peptides led to a significant reduction in cell survival relative to
the control cells (p < 0.0001). Incubation of the PC12
and SH-SY5Y cells with PT (open bars), suramin
(diagonally striped bars), and GDP S (cross-hatched
bars) protected them from amyloid-induced cell death relative to
untreated cells exposed to the amyloids (solid bars).
Incubation of the cells with inhibitors in the absence of the amyloid
peptides had no significant effect on cell viability (Control
Cells). * and ** indicate that the increases in cell viability
relative to the untreated cells exposed to the amyloids were
significant at p < 0.0005 and p < 0.0001, respectively.
|
|
 |
DISCUSSION |
In previous cell culture studies, the most important predictor of
A
toxicity was the macromolecular state of the A
peptides with
only aged or aggregated A
peptides (including fibrils, protofibrils, and/or low molecular weight intermediates) consistently eliciting toxic
responses (12-18). Additionally, both the L- and
D-enantiomers of A
exhibited nearly identical structural
characteristics and induced similar levels of toxicity, implying that
A
neurotoxicity was mediated by A
fibril features instead of any
stereoisomer-specific interactions (79). Compounds such as Congo Red,
rifampicin, and recognition peptides that bind to and/or inhibit the
formation of amyloid fibrils have also been shown to attenuate the
toxicity of A
, further establishing the causal link between
A
structure and function (21-24, 64, 80, 81).
Since the neurotoxic effects of A
peptides appear to be linked to
peptide structures associated with aggregation and amyloid fibril
formation, any plausible molecular mechanism of A
toxicity should
also demonstrate structural dependence. Our cell membrane and purified
subunit data, which suggest that the A
toxicity is mediated through
a pathway involving peptide-induced G protein activation, possess the
required structural specificity. We found that the GTPase activities
associated with PC12 membranes and G
o and
G
i vesicles only increased with exposure to the peptides containing extensive
-sheet and amyloid contents, A
-(1-40) and A
-(25-35). The nontoxic and nonaggregated A
-(1-16) produced no
discernible effects on the GTPase activities of the PC12 membranes or
the G
o and G
i vesicles.
Ample evidence exists that suggests that G protein activation and other
signal transduction events such as phospholipase D and adenylate
cyclase activation may be associated with the biological activity of
A
(82-84). Also, the reported changes in K+ and
Ca2+ ion channel activity and changes in calcium
homeostasis are all consistent with GTPase activation being an early
event in the mechanism of action of A
(26, 30, 85, 86). However, in very few of these studies has anyone shown the relationship between the
biological activity and the structure of the peptide, which is
essential in establishing the connection between activity and a
toxicity mechanism.
Unlike our results, reports of A
-induced barium conductances in
N1E-115 neuroblastoma cells (87) and calcium fluxes (85, 88) did not
correlate aggregation state of the peptide with activity. When A
blockage of the fast-inactivating K+ current was
investigated, structure-function relationships were examined, but no
structure dependence upon ion channel activity was observed (30). In
addition, the data associated with the free radical model of A
toxicity such as the ability of A
peptides to generate EPR signals
were not structure-specific (31).
We suggest that amyloidogenic A
-induced G protein activation could
be an early step in the molecular level mechanism of A
toxicity and
that activation of alternative G protein pathways could produce many of
the observed diverse cellular responses. For example, in our studies,
PT had significant inhibitory effects on GTPase activity and toxicity,
indicating that the Gi and Go families of G
proteins were being activated (74-78). The results of our purified
G
o and G
i subunit GTPase assays confirm
this hypothesis. The amyloidogenic A
-(1-40) and A
-(25-35)
significantly increased the GTPase activity of both the
G
o and G
i subunits. Activation of these
particular families of G proteins could account for some of the
previous ion channel results because they have been linked to certain
K+ and Ca2+ channel modulations (89-93). Our
results do not preclude the activation of multiple GTPases, which could
result in tremendously diverse intracellular phenomena since G proteins
and their effectors have been associated with selective protein
phosphorylation, gene transcription, cytoskeletal reorganization,
secretion, and membrane depolarization (71, 74, 92, 94-96).
In both our Pronase experiments and in our G
o and
G
i subunit experiments, we demonstrated that the
presence of receptors was not necessary for the observed amyloidogenic
peptide-induced increases in GTPase activity. In the Pronase-treated
cells, few receptors remained, as evidenced by the absence of an
epinephrine-induced increase in GTP hydrolysis. Similarly, no receptors
were included in the G
o and G
i subunit
preparations. However, in both systems, significant peptide-induced
increases in GTPase activity were observed. Nonreceptor mediated GTPase
activation has been documented with mastoparan, ethanol, and shear
stress (59, 97-99). The discovery that our A
GTPase activities may
be membrane-mediated is consistent with a number of other previous
findings, which imply the importance of A
-membrane interactions to
neurotoxicity. A
has been shown to interact with the lipid bilayer
of the plasma membrane, forming cation-selective channels and
disrupting ion homeostasis (25, 26). Interaction with the plasma
membrane may also influence aggregation and amyloid formation of the
peptide (33, 34, 100, 101).
In conjunction with our A
GTPase results, our calcitonin results
suggest that the amyloidoses may share some common steps in the
mechanism of toxicity. Analogous to our A
findings, we found that
amyloidogenic bovine calcitonin in water containing 5 mM
CaCl2 and 1 mM MgCl2 increased cell
toxicity and increased GTPase activities associated with PC12 membranes
and G
o and G
i vesicles, whereas the
nonamyloidogenic calcitonin in deionized water did not significantly
alter cell viability or G protein activities of PC12 membranes or
G
o and G
i vesicles. Amyloidogenic bovine
calcitonin increased the GTPase activity in Pronase-treated cells and
G
o and G
i vesicles devoid of receptors,
and inhibition of GTPase activation attenuated cell toxicity. These
results again suggest the potential importance of protein-membrane
interactions to amyloid-mediated toxicity and indicate that GTPase
activation is an early step in the toxicity of amyloid-forming peptides.
In conclusion, we demonstrated that A
and calcitonin peptides
altered G protein activity in a structure-specific manner; only the
peptides with extensive
-sheet and amyloid contents significantly
increased GTPase activity. Both Go and Gi
activation was observed. At least some of the observed amyloid-induced
increases in GTPase activity were not receptor-mediated, pointing to
the potential importance of peptide-membrane interactions in the
biological activity of the peptides. We demonstrated that the observed
increases in G protein activity were linked to neurotoxicity by showing that G protein inhibitors significantly reduced the neurotoxic effects
of the amyloidogenic A
and calcitonin peptides. These results may
help to elucidate the mechanism of toxicity and may lead to novel
treatments for the 16 or more diseases associated with amyloid proteins.