From the Laboratory of Persistent Viral Diseases,
Rocky Mountain Laboratories, NIAID, National Institutes of Health,
Hamilton, Montana 59840 and the § Department of Veterinary
Public Health and the National Research Center for Protozoan Diseases,
Obihiro University of Agriculture and Veterinary Medicine,
Obihiro Hokkaido 080-8555 Japan
Received for publication, January 12, 2001, and in revised form, January 29, 2001
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ABSTRACT |
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The formation of protease-resistant prion protein
(PrP-res or PrPSc) involves selective interactions
between PrP-res and its normal protease-sensitive counterpart, PrP-sen
or PrPC. Previous studies have shown that synthetic peptide
fragments of the PrP sequence corresponding to residues 119-136 of
hamster PrP (Ha119-136) can selectively block PrP-res formation in
cell-free systems and scrapie-infected tissue culture cells. Here we
show that two other peptides corresponding to residues 166-179
(Ha166-179) and 200-223 (Ha200-223) also potently inhibit the
PrP-res induced cell-free conversion of PrP-sen to the
protease-resistant state. In contrast, Ha121-141, Ha180-199, and
Ha218-232 were much less effective as inhibitors. Mechanistic analyses
indicated that Ha166-179, Ha200-223, and peptides containing residues
119-136 inhibit primarily by binding to PrP-sen and blocking its
binding to PrP-res. Circular dichroism analyses indicated that
Ha117-141 and Ha200-223, but not non-inhibitory peptides, readily
formed high Transmissible spongiform encephalopathies
(TSEs)1 are a group of fatal
neurodegenerative diseases that include scrapie in sheep and goats,
bovine spongiform encephalopathy in cattle, chronic wasting disease in
deer and elk, and Creutzfeldt-Jakob disease in humans. The pre-eminent
neuropathological feature of TSEs is the accumulation of the
disease-specific, protease-resistant isoform of prion protein,
designated as PrP-res or PrPSc, in the central nervous
system. PrP-res is generated post-translationally from the normal,
protease-sensitive isoform of prion protein, PrP-sen or
PrPC. Although several lines of evidence suggest that
PrP-res is a major component of the infectious TSE agent, the full
identity of the agent is still unclear.
Studies using transgenic mice, PrP-deficient mice, and neuronal tissue
grafts revealed that an interaction between PrP-sen and PrP-res that
leads to PrP-res accumulation plays a central role in the propagation
of infectivity and neurodegeneration (1-3). Furthermore, the formation
of PrP-res and propagation of TSE infectivity in animals and cultured
cells require PrP amino acid sequence compatibility between the
recipient and donor of infectivity (4-6). Cell-free experiments have
shown that PrP-res can selectively bind PrP-sen (7, 8) and subsequently
induce its conversion into a PrP-res-like protease-resistant molecule
(9). Further studies of this two-step process revealed that the direct
binding of PrP-sen to PrP-res is less dependent upon PrP amino acid
compatibility than the conformational transformation to the
protease-resistant state (10, 11). Although previous efforts have
described several aspects of the interaction between the two PrP
isoforms, the molecular details of the interaction and conversion
mechanism remain to be elucidated.
Direct interactions of synthetic PrP peptides with PrP molecules
indicated the possible usefulness of PrP synthetic peptides for
studying the mechanism of PrP-sen-PrP-res interaction. For instance,
Ha109-141 and Ha119-136 can inhibit PrP-res formation in cell-free
PrP-res-induced conversion reactions and in scrapie-infected cell
cultures (12, 13). Stoichiometric excesses of a synthetic peptide
corresponding to hamster PrP residues 90-145 (Ha90-145) can bind
PrP-sen and protect it from proteolysis (14, 15). The central region of
PrP containing residues 90-145 has been of prime interest because it
is part of the usual C-terminal protease-resistant core of PrP-res and
it contains an amyloidogenic sequence AGAAAAGA that appears critical
for PrP-res formation (12, 13, 15-18).
Other data suggest that other regions in the C-terminal half of the
molecule might be important in PrP-sen-PrP-res interactions leading to
PrP-res formation (8, 19-21). In this study, we have investigated this
possibility further by testing the effects of various synthetic
peptides corresponding to portions of the C-terminal half of the PrP
amino acid sequence. Ha166-179 and Ha200-223 strongly inhibited the
protease-resistant PrP formation in cell-free conversion assays, while
Ha180-199 showed much weaker inhibition. In addition, we have
addressed mechanistic aspects of the inhibition by various peptides,
including Ha109-141, and found that the peptides inhibit by binding to
PrP-sen and blocking its binding to PrP-res.
Cells and Purification of PrP-sen--
Hamster PrP-sen lacking
the glycosylphosphatidylinositol (GPI) anchor was expressed in PA317
and psi2 mouse fibroblasts (9). We refer to the PrP-sen lacking a GPI
anchor as PrP-sen(GPINEG). Metabolic labeling of the cells
with [35S]methionine and purification of
35S-labeled PrP-sen were performed as described previously
(22) except for the elution of 35S-labeled PrP-sen from
protein A-Sepharose immunocomplexes; PrP-sen was eluted with 0.1 N acetic acid (pH 2.8) and kept at 4 °C until use. In
some experiments, culture supernatants of 35S-labeled cells
were used as a source of
35S-PrP-sen(GPINEG).
Synthetic Peptides--
Synthetic peptides of hamster PrP
corresponding to residues 109-141 (Ha109-141,
MKHMAGAAAAGAVVGGLGGYMLGSAMSRPMMHF), residues 117-141 (Ha117-141,
AAGAVVGGLGGYMLGSAMSRPMMHF), residues 121-141 (Ha121-141,
VVGGLGGYMLGSAMSRPMMHF), and residues 218-232 (Ha218-232, YQKESQAYYDGRRSS) were described previously (12). In the experiments of
the present study, Ha109-141 and Ha117-141 were sometimes used as
surrogates for one another since they have been shown to be similarly
inhibitory and both contain the core inhibitory sequence of residues
119-136 (13). Synthetic peptides of hamster PrP corresponding to
residues 166-179 (Ha166-179, VDQYNNQNNFVHDC), residues 180-199
(Ha180-199, VNITIKQHTVTTTTKGENFTC), and residues 200-223 (Ha200-223,
ETDIKIMERVVEQMCTTQYQKESQ) were synthesized in this study (Fig.
1). Randomized peptides of Ha166-179
(RHa166-179, VNFQNDVHNYQDNC) and Ha200-223 (RHa200-223a,
MIQETMKVTDQSIECQEVTKQRYE, and RHa200-223b, SQEKQYQTTCMQEVVREMIKIDTE)
were also synthesized. Synthetic peptides Ha109-141, Ha117-141,
Ha121-141, Ha180-199, Ha200-223, and Ha218-232 were dissolved with
deionized water to make 2 mM stock solutions. Due to poorer
solubility in water, synthetic peptides Ha166-179, RHa166-179, and
RHa200-223a were first dissolved in dimethyl sulfoxide to make
10 mM stock solutions. Aliquots of each stock solution were
stored at Cell-free Conversion Reactions--
PrP-res was purified without
proteinase K (PK) treatment from the brains of hamsters infected with
the 263K strain (23) as described previously (24). Cell-free conversion
reactions without the use of GdnHCl were performed as described
elsewhere (8). Briefly, purified PrP-res was diluted with deionized
water and sonicated for 15 s, and then 100 ng of PrP-res was mixed
with 20,000 cpm (~2 ng) of
35S-PrP-sen(GPINEG) in 20 µl of reaction
mixture containing 200 mM KCl, 1.25% Sarkosyl, 5 mM MgCl2, and 50 mM citrate buffer
(pH 6.0). The reaction mixtures were incubated at 37 °C for 2 days.
Nine-tenths of the reaction mixtures were treated with 20 µg/ml PK
(50 mM Tris-HCl (pH 8.0), 150 mM NaCl in 100 µl) at 37 °C for 30 min. PK digestion was stopped by adding
Pefabloc (Roche Molecular Biochemicals) to 2 mM,
thyroglobulin to 200 µg/ml as a carrier protein, and 4 volumes of
methanol. The remaining one-tenth of the reaction mixture was analyzed
without PK treatment. The protein samples collected by centrifugation were subjected to SDS-PAGE using Novex pre-cast acrylamide gels and
radioactive proteins were visualized and quantified with a PhosphorImager instrument (Molecular Dynamics).
PrP-sen/PrP-res Binding Analysis and Conversion Reactions in
Multiwell Plates--
PrP-res was suspended in 2.5 M
GdnHCl at 2.5 ng/µl and incubated at 37 °C for 30 min. Then 40 µl of the PrP-res solution was added to the round-bottom 96-well
plate (High bind, Costar) and incubated at 37 °C overnight. After
adsorption of PrP-res, the wells were washed once with
phosphate-buffered saline and then blocked with 0.5% skim milk in
phosphate-buffered saline at room temperature for 2 h. After
washing the wells once with phosphate-buffered saline, 40 µl of
35S-PrP-sen(GPINEG) (40,000 cpm) solution
containing 200 mM KCl, 5 mM MgCl2,
1.25% Sarkosyl, and 0.1% fetal bovine serum in 50 mM
citrate buffer (pH 6.0) was added and the plates were incubated at
37 °C for 2 days. For the binding analysis, the wells were washed
four times with a washing buffer containing 200 mM KCl,
1.25% Sarkosyl, and 50 mM citrate buffer (pH 6.0). For the
conversion reactions, the wells were washed once with 50 mM
Tris-HCl (pH 8.0) and 150 mM NaCl, and then incubated with
50 µl of PK solution containing 20 µg/ml PK, 50 mM
Tris-HCl (pH 8.0), and 150 mM NaCl at 37 °C for 30 min.
The PK digestion was terminated by adding 2 µl of 0.1 M
Pefabloc and then the wells were washed once with 50 mM Tris-HCl and 150 mM NaCl. Finally, 35S-PrP
remaining in the wells was eluted with 20 µl of 1 × sample buffer (5% SDS, 4 M urea, 62.5 mM Tris-HCl (pH
6.8), 3 mM EDTA, 5% glycerol, 5% 2-mercaptoethanol,
0.04% bromphenol blue) by heating the plate at 80 °C for 10 min and
analyzed by SDS-PAGE as above.
Binding Analysis between PrP-sen and PrP Synthetic
Peptides--
Peptides were diluted to various concentrations with 50 mM phosphate buffer either (pH 8.5 or pH 7.0) containing
150 mM NaCl and 50 µl of the diluted peptide solutions
were added to the wells of a DNA-BIND 96-well plate (Costar). The wells
were coated with a layer of reactive N-oxysuccinimide esters
so that peptides could be covalently cross-linked to the wells through
a primary amine group. After 90 min incubation at 37 °C, the wells
were washed once with the corresponding buffer and blocked with 2%
skim milk in the corresponding buffer. The wells were washed once with
phosphate-buffered saline and then incubated with 40 µl of
35S-PrP-sen(GPINEG) (40,000 cpm) solution
containing 200 mM KCl, 5 mM MgCl2,
1.25% Sarkosyl, and 0.2% fetal bovine serum in 50 mM
citrate buffer (pH 6.0) at 37 °C overnight. After incubation,
supernatants were saved as the unbound fraction, and the wells were
washed four times with washing buffer. Finally, the
35S-PrP-sen bound to the wells (bound fraction) was eluted
with 5% SDS and 4 M urea by heating the plate at 80 °C
for 10 min, and radioactivity in the bound and unbound fractions was
counted by liquid scintillation counter. To analyze the selectivity of the binding between PrP-sen and peptides, the culture supernatants of
35S-labeled cells were used instead of
35S-labeled purified PrP-sen. In this case, unbound and
bound fractions were analyzed by SDS-PAGE.
Immunoblotting--
Transfer of the proteins from acrylamide
gels to Immobilon-P membranes (Millipore) was performed as described
elsewhere (25). PrP on the membrane was visualized by using anti-PrP
synthetic peptide (residues 89-103) antibodies and ECF Western
blotting reagents (Amersham Pharmacia Biotech), and quantified with a
PhosphorImager instrument.
Circular Dichroism Spectroscopy--
Synthetic peptides were
dissolved in deionized water to make 5 mg/ml stock solutions. Peptide
solutions (40 µl) were combined with 160 µl of conversion buffer
(200 mM KCl, 5 mM MgCl2, 50 mM citrate, pH 6.0) with or without 0.1% Sarkosyl to give
final peptide concentrations of 1 mg/ml. CD measurements were performed
using a 0.1-mm path length quartz cylindrical cell with an OLIS-16 DSM CD Spectrophotometer. The following parameters were used: 0.05 nm
monochromator resolution, 1 nm band width, dual beam mode, 400 kHz
sampling rate, 2 V high volts criterion. Wavelength calibration was
done with (+)-(1S)-10-camphorsulfonic acid (Sigma). The
temperature of sample chamber was held at 37 °C. Data were collected
from 260 to 190 nm with 1 datum/nm. The resulting spectra were obtained by averaging 6 scans and subtracting spectra of the buffer with or
without 0.1% Sarkosyl.
Inhibition of Protease-resistant PrP Formation in Cell-free
Reactions by Synthetic PrP Peptides--
Previous cell-free conversion
studies have shown that synthetic peptides Ha106-128, Ha109-141, and
certain subunits thereof (e.g. Ha117-121) can inhibit
PrP-res formation, while peptides spanning most other portions of the
PrP sequence do not (12, 13). One portion of the PrP amino acid
sequence that was not addressed in previous analyses spanned residues
170-218. Thus, we first examined whether PrP synthetic peptides from
this region inhibit the PrP conversion reaction. Cell-free PrP
conversion reactions usually involve incubating immunoprecipitated
[35S]methionine-labeled PrP-sen with unlabeled PrP-res
purified from TSE-infected brain tissue and then assaying for the
formation of partially proteinase K (PK)-resistant 35S-PrP
products (26). In the previous peptide inhibition studies, the
cell-free conversion reactions were performed using GdnHCl as a
stimulant (12, 13). However, we recently established cell-free
conversion reactions that occur under much more physiologically compatible conditions without the use of chaotropic salts (8). Hence,
to better approximate in vivo PrP-res formation, we used these conditions in the experiments that follow.
As reported previously using the GdnHCl-containing conditions (12),
Ha117-141 inhibited the formation of the typical partially PK-resistant 35S-PrP conversion product. Like brain-derived
PrP-res itself (not shown), these 35S-PrP conversion
product bands appear to be ~7 kDa lower in molecular mass than the
full-length 35S-PrP-sen precursor after PK treatment (Fig.
2a). The concentration of the
peptide exhibiting 50% inhibition of the conversion reaction (the
IC50) was ~40 µM (Fig. 2g)
compared with 80 µM observed under the GdnHCl-containing
conditions in the previous report (12). As in the previous study, the
peptides Ha121-141 and Ha218-232 did not inhibit the
protease-resistant PrP formation at concentrations up to 500 µM (Fig. 2, b and f). Thus, other
than the somewhat lower IC50 for Ha117-141, the effects of
these peptides were similar in GdnHCl-containing and GdnHCl-free
cell-free conversion reaction conditions.
Two newly synthesized peptides Ha166-179 and Ha200-223 inhibited
protease-resistant PrP formation with IC50 values of 10-15 µM (Figs. 2, c, e, and g). The
inhibitory effects of these peptides are at least comparable to that of
the synthetic peptide Ha109-141 under the present conditions
(IC50 = 15), which was the most efficient inhibitory
peptide in previous studies (data not shown). In contrast, the
synthetic peptide Ha180-199 only partially inhibited
protease-resistant PrP formation at the highest concentration tested
(500 µM) (Fig. 2, d and g).
Since our data demonstrate the strongest inhibition by Ha166-179 and
Ha200-223, we wished to address the amino acid sequence specificity of
the effects of these peptides. Thus, we synthesized and examined
peptides of the same amino acid composition, but randomized sequence
(RHa166-179, RHa200-223a, and RHa200-223b, respectively) for effects
on the conversion reaction. RHa166-179 did not inhibit the reaction up
to 500 µM, demonstrating the amino acid sequence
specificity of the Ha166-179 effect (Fig.
3a). However, two distinct
RHa200-223 peptides (data for only one is shown in Fig. 3b)
substantially inhibited the reaction at the highest concentration tested (500 µM) indicating that the effect of the
Ha200-223 was not completely dependent upon the full amino acid
sequence of the peptide.
Inhibition of PrP-sen/PrP-res Binding by Synthetic
Peptides--
Since some of the PrP synthetic peptides inhibited the
formation of protease-resistant 35S-PrP, we examined
whether this was due to the inhibition of the binding between PrP-sen
and PrP-res or the subsequent conversion of PrP-sen to the
protease-resistant form. PrP-sen is soluble and PrP-res is a readily
pelletable aggregate. Thus, we first attempted to examine the effect of
the peptides on PrP-res binding by using a sedimentation-based binding
assay described previously (8, 11). However, 35S-PrP-sen
was detected in the pellet in the presence of some of the peptides
without PrP-res (data not shown) thus hampering our ability to
specifically monitor binding to PrP-res. Therefore, we opted to monitor
both the PrP-sen-PrP-res binding and conversion reactions using a solid
phase system with PrP-res adsorbed to a 96-well plate.
Fig. 4 shows the binding and the
conversion reaction with the solid phase system. In the absence of
pre-adsorbed PrP-res, the nonspecific binding of
35S-PrP-sen to the wells was minimal and no
protease-resistant 35S-PrP was detected after the PK
digestion. However, in the presence of PrP-res, both binding of
35S-PrP-sen and conversion to the PK-resistant form was
detected (Fig. 4, right side lanes). Another indication of
the specificity of the solid phase binding and conversion reactions was
inhibition by anti-PrP219-232, a rabbit antiserum known to inhibit the
binding of PrP-sen to PrP-res in suspension reactions (8), but not by
normal rabbit serum. Given these indications of the specificity of the
solid phase system, we used it in subsequent analyses of the effects of
the peptide inhibitors.
Synthetic peptides Ha109-141, Ha166-179, and Ha200-223, which
inhibited the formation of protease-resistant 35S-PrP, also
blocked the binding of 35S-PrP-sen to PrP-res in a
dose-dependent manner (Fig.
5). In contrast, Ha121-141 and
Ha218-232, which did not inhibit the conversion reaction, did not
block PrP-sen-PrP-res binding. Furthermore, Ha180-199, which only
partially inhibited the conversion reaction at 500 µM,
did not significantly block the binding up to 100 µM. Therefore, these results indicated that the inhibition of
protease-resistant PrP formation by Ha109-141, Ha166-179, and
Ha200-223 was due primarily to a blockade of the binding of PrP-sen to
PrP-res.
Peptide Inhibition by Binding to PrP-sen--
The preceding
results raise the question of whether the inhibition of PrP-sen-PrP-res
binding by the peptide inhibitors is due to binding of the synthetic
peptide to PrP-sen, PrP-res, or both. In a solid phase analysis, in
which the synthetic peptides were covalently cross-linked to a 96-well
plate, binding of 35S-PrP-sen to Ha117-141, Ha166-179,
and Ha180-199 was observed (Fig. 6). In
contrast, no binding of 35S-PrP-sen to Ha121-141,
Ha200-223, and Ha218-232 was detected. This experiment was performed
with the multiwell plate coated with N-oxysuccinimide that
is reactive with primary amines (DNA-BindTM, Costar).
Moreover, the same results were obtained when the plate coated with a
sulfhydryl-reactive maleimide group (Reacti-BindTM,
maleimide-activated plate, Pierce) or a plate possessing a hydrophobic surface (High bind, Costar) was used (data not shown). One apparent inconsistency in our data was observed with Ha200-223; this peptide inhibited the binding of PrP-sen to PrP-res (Fig. 5) but binding of
PrP-sen to this peptide was not detected by the solid phase assay.
However, when sedimentation analysis was performed,
35S-PrP-sen was detected in the pellet with Ha200-223 even
without PrP-res (data not shown). We also observed by electron
microscopy that Ha200-223 forms fibrils under these buffer conditions
(data not shown). These findings suggest that PrP-sen was bound to
Ha200-223 fibrils in suspension and was therefore pelleted by
centrifugation. Taken together, the solid phase and sedimentation
experiments provide evidence for PrP-sen binding to each of the
inhibitory peptides.
Next we tested if the peptide can also inhibit the PrP-sen-PrP-res
interaction by binding to PrP-res. For this purpose, the synthetic
peptides and 35S-PrP-sen were added sequentially to the
wells coated with PrP-res. The synthetic peptide was added first and
incubated for 1 day to allow binding to PrP-res. Then the peptide
solution was removed and 35S-PrP-sen was added. No peptides
inhibited binding between 35S-PrP-sen and PrP-res under
these circumstances (data not shown) indicating that the inhibitory
peptides do not interact with PrP-res in a manner which blocks PrP-sen
binding after removal of the free peptide solution and the addition of
PrP-sen. Thus, the available evidence favors a mechanism in which the
inhibitory peptides act by binding to PrP-sen and blocking its binding
to PrP-res.
Specificity of the Binding between Peptide Inhibitors and
PrP-sen--
To examine the specificity of the binding between the
peptide inhibitors and PrP-sen, selectivity of the binding of PrP-sen versus other proteins was analyzed (Fig.
7). When culture supernatants of
[35S]methionine-labeled cells containing many different
labeled proteins besides PrP-sen(GPINEG) were incubated in
wells coated with PrP-res, the binding between PrP-sen and PrP-res was
highly selective as described previously (8); the binding of only the
three major PrP-sen(GPINEG) bands was detected
(indicated by arrowheads). The identity of these bands as
35S-PrP-sen was confirmed by the ~80% decrease in
binding when PrP-sen(GPINEG) was first depleted from the
culture supernatant by immunoprecipitation. The binding of
35S-PrP-sen(GPINEG), as well as some other
35S-labeled proteins, to PrP synthetic peptides was
observed in wells coated with Ha109-141, Ha166-179, or Ha180-199.
This was consistent with the peptide-PrP-sen binding analysis using
purified 35S-PrP-sen (Fig. 6). The depletion of
PrP-sen(GPINEG) reduced the intensity of only the
PrP-sen(GPINEG) bands (indicated by arrowheads),
suggesting that the other 35S-labeled bands were not
SDS-stable oligomers of PrP-sen but other 35S-labeled
proteins in the culture supernatants. Although the binding between
PrP-sen and PrP synthetic peptides was not as selective as that between
PrP-sen and PrP-res, this result confirmed that Ha109-141, Ha166-179,
and Ha180-199 bind PrP-sen in preference to a large number of other
proteins.
Secondary Structures of the Inhibitory Peptides--
Inhibitory
peptides described by Chabry et al. (12) tended to form high
Since TSE pathogenesis and PrP-res formation involve precise
interactions between PrP isoforms, we have studied the effects of
synthetic PrP peptides to gain insight into these interactions. Previous studies revealed that Ha109-141 and shorter subfragments such
as Ha117-141 inhibited the transformation of PrP-sen to
protease-resistant forms in vitro (12, 13). In this study,
we identify peptides from three other segments within the C-terminal
third of the PrP sequence that inhibit protease-resistant PrP
formation. Ha166-179 and Ha200-223 have potencies (IC50
values) that are comparable to that of Ha109-141 and Ha117-141, while
Ha180-199 was much less potent. There was strong amino acid sequence
dependence to the inhibition by Ha166-179, but this dependence was
less pronounced for Ha200-223.
It is unclear why two different randomized permutations of Ha200-223
inhibited the PrP-res formation to some extent. However, it is possible
that a certain oligopeptide or structural motif was maintained in the
randomized sequences that allowed binding to PrP-sen or PrP-res and
inhibition of conversion. Although we could not discern any obviously
conserved pattern of residues on cursory examination, identification of
such a motif may contribute to the understanding of the molecular
mechanism of PrP-res formation and possible therapeutics for TSEs.
The PrP-res-induced transformation of PrP-sen into
protease-resistant forms can be separated kinetically and biochemically into two different steps; first, the binding between PrP-sen and PrP-res, and second, the conversion of the bound PrP-sen to PrP-res (7,
8, 11). Thus, the generation of protease-resistant PrP can be impaired
either at the binding or conversion steps. Our data indicate that the
inhibition of protease-resistant PrP formation by the PrP peptides
Ha109-141, Ha166-179, and Ha200-223 was due to interference with the
initial binding between the two PrP isoforms. Since Ha117-141 and
Ha166-179 were able to bind PrP-sen, but were not inhibitory when
preincubated with PrP-res, it is likely that their inhibition of
conversion is due to binding to PrP-sen in a way that hinders the
PrP-sen-PrP-res interaction. Interestingly, Ha180-199 was more
efficient than Ha200-223 at binding of PrP-sen in the solid phase
assay, but was 5-10-fold less potent as an inhibitor of conversion.
Hence, although we conclude that inhibition of PrP conversion by
Ha109-141, Ha166-179, and Ha200-223 is likely to be due to peptide
binding to PrP-sen, the binding of peptides to PrP-sen is not always
sufficient to block conversion. Further analyses will be required to
clarify whether or not direct binding of the peptides to PrP-res occurs.
In the cases of Ha109-141, Ha117-141, and Ha166-179, where binding
to PrP-sen appears to be important for inhibition, at least a couple of
different mechanisms can be envisioned to account for these effects.
First, the peptides, or polymers thereof, might bind to PrP-sen and
physically block the PrP-res-binding site. Alternatively, the binding
of the peptides might overstabilize PrP-sen or cause a change to a
conformation that is incapable of binding PrP-res.
For mechanistic reasons, it is important to consider the stoichiometry
of the inhibitory peptides relative to PrP-sen and PrP-res in the
conversion reactions. The cell-free conversion reactions contained ~2
nM PrP-sen(GPINEG) and ~150 nM
PrP-res. Thus, the peptides with IC50 values of The direct binding of PrP-sen to Ha109-141, Ha166-179, and
(apparently) Ha200-223, but not numerous other PrP-derived peptides, suggests that the PrP-sen-binding domain on PrP-res may include residues contained in these inhibitory peptides. The lack of observed binding between PrP-sen and Ha200-223 in the solid phase analysis may
be due to a lack of Ha200-223 fibril formation or some other artifactual interference with PrP-sen binding on the solid phase support. It is noteworthy that Ha200-223, unlike the other peptides, has internal lysine and cysteine residues that could react to the
derivatized wells. This might restrict interactions with PrP-sen more
than binding solely via N- or C-terminal residues which is the mode of
attachment for the other peptides. Interestingly, a previous analysis
has suggested that a specific antibody that inhibits the binding and
conversion reactions would likely sterically hinder access to residues
on PrP-sen that are contained in the peptides Ha109-141, Ha166-179,
and Ha200-223 (8). Thus, the available data suggest that residues
contained in one or more of these three inhibitory peptides may be
important in the binding sites on both PrP-sen and PrP-res.
Dimerization of PrP molecules is proposed to generate a PrP-res
specific epitope (28), suggesting the possibility that a conformational
domain comprising several regions of one PrP-res molecule or several
regions of different PrP-res molecules is involved in binding to
PrP-sen and inducing its conformational transformation. It is
conceivable that these regions on the PrP-res molecule correspond to
the inhibitory peptides identified in this and previous studies.
The binding of PrP-sen to the individual PrP peptide inhibitors was not
adequate to form protease-resistant PrP even at the highest
concentration tested (Fig. 2), consistent with the idea that multiple
regions on PrP-res are involved in causing the conformational transformation of PrP-sen. As noted above, a previous study showed that
vast stoichiometric excesses of Ha90-145 could cause PrP-sen to gain
some PK-resistance, however, this was not the characteristic partial
PK-resistance of TSE-associated PrP-res (14). Thus, it is not yet
apparent that any synthetic peptide can induce the conversion of
PrP-sen to bona fide PrP-res. The conformation of the
peptide inhibitors may have similarity but not identity to the
corresponding PrP-sen-binding domain on PrP-res polymers. Thus
incomplete reconstitution of the PrP-sen-binding domain by individual
peptides may also account for lower selectivity than PrP-res in the
binding of PrP-sen versus other proteins (Fig. 8). Perhaps a
combination of different PrP synthetic peptides in a given spatial
distribution will be required to more closely mimic PrP-sen-PrP-res
interactions. However, initial attempts at combining Ha166-179 and
Ha200-223 at 2.5 and 5 µM, respectively (i.e.
slightly below their IC50 values), did not indicate either additive or synergistic effects of these two peptide inhibitors (data
not shown).
Our data suggest that the region including residues 166-179, which
forms an extended loop structure between the second Compounds that can facilitate the clearance of accumulated PrP-res
(34), stabilize PrP-sen, or prevent interactions between the two PrP
isoforms, are possible candidates for TSE therapeutics (12, 13,
35-37). In addition, compounds indirectly affecting PrP-res formation
may also have therapeutic value. The combined usage of compounds
with different mechanisms for inhibiting of PrP-res accumulation may
have synergistic effects in TSE therapeutics. The list of compounds
which inhibit PrP-res formation and/or prolong the incubation periods
of scrapie in rodents is growing (35, 37-40). It is important to know
the mechanism of action of these compounds. The experimental procedures
used in this study may contribute to the analysis of such mechanisms.
In addition, the assays of the direct interaction between PrP-sen and
PrP-res and/or PrP synthetic peptides in multiwell plates may provide
high throughput screens for compounds that inhibit those interactions.
-sheet structures when placed under the conditions of
the conversion reaction. We conclude that these inhibitory peptides may
mimic contact surfaces between PrP-res and PrP-sen and thereby serve as
models of potential therapeutic agents for transmissible spongiform encephalopathies.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
20 °C until use.
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Fig. 1.
Schematic representation of the location of
PrP synthetic peptides. The top line with
boxes indicates HaPrP23-232 which corresponds to the
sequence of the mature PrP-sen which lacks the N- and C-terminal signal
sequences. Open boxes indicate regions forming -strands
(
1 and
2) and hatched boxes indicate regions forming
-helix (
1,
2, and
3) according to NMR studies (29, 41). The
thick bars with associated residue numbers indicate the
regions spanned by synthetic peptides used in this study. In some
experiments a peptide corresponding to residues 117-141 (not
illustrated) was also used.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 2.
The effect of PrP synthetic peptides on the
cell-free formation of protease-resistant PrP. a,
Ha117-141; b, Ha121-141; c, Ha166-179;
d, Ha180-199; e, Ha200-223; f,
Ha218-232. The lanes labeled
PrP-sen(GPINEG) show one-tenth
equivalent of 35S-labeled PrP-sen(GPINEG) used
for the cell-free conversion reactions. Peptide concentrations
(µM) are indicated above the gel images.
+PK indicates the samples treated with PK. The presence and
absence of PrP-res in the reaction are indicated by "+" and
" ," respectively. The upper and lower
brackets on the left indicate PK-untreated
35S-PrP-sen(GPINEG) and PK-resistant
35S-PrP, respectively, that were used in the phosphor
autoradiographic quantitation shown in (g). Conversion efficiencies
(mean % conversion ± S.D. (n = 3-5)) were
calculated relative to a control reaction without peptide as described
previously (8). Molecular mass markers (kDa) are on the
right.
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Fig. 3.
Effects of randomized permutations of
Ha166-179 (RHa166-179) (a) and Ha200-223
(RHa200-223a) (b) on the formation of
protease-resistant PrP. Details other than the peptides used are
the same as described in the legend of Fig. 2.
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Fig. 4.
Solid phase binding and conversion
reactions. 35S-PrP-sen(GPINEG) was
incubated for 2 days in 96-well plates with or without precoating with
PrP-res. The wells were washed and incubated with (+) or without ( )
PK. 35S-PrP species bound to the wells were solubilized and
analyzed by SDS-PAGE and phosphor autoradiography. Specificity of the
reaction was tested by comparing the effects of inclusion of
219-232, an antiserum known to inhibit binding of
35S-PrP- sen(GPINEG) to PrP-res, and normal
rabbit serum (NRS). The brackets are
as described in legend to Fig. 2. Abs, antibodies.
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Fig. 5.
Effects of synthetic PrP peptides on
binding of 35S-PrP- sen(GPINEG) to
immobilized PrP-res. Using the solid phase binding assay used in
Fig. 4, peptides were added in increasing concentrations to the binding
buffer with 35S-PrP-sen(GPINEG). After
incubation for 2 days, the wells were washed and assayed for bound
35S-PrP(GPINEG). The data points indicate
mean ± S.D. (n = 3-4) normalized to
radioactivity bound in the absence of any peptide. The graphs are
labeled with the span of HaPrP residues to which the peptides
correspond.
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Fig. 6.
Binding of
35S-PrP- sen(GPINEG) to covalently
immobilized PrP peptides.
35S-PrP-sen(GPINEG) (40,000 cpm) was added to
wells pre-coated with PrP synthetic peptides as described under
"Experimental Procedures." Supernatants were saved as the unbound
fraction and the 35S-PrP(GPINEG) eluted
from the wells with 5% SDS and 4 M urea by heating at
80 °C was designated the bound fraction. The graph shows
mean ± S.D. (n = 3) of the percentage of the
bound 35S-PrP(GPINEG) against the total
35S-PrP(GPINEG) in the reaction (the sum of the
unbound and bound fractions).
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Fig. 7.
Selectivity of the binding between PrP-sen
and PrP synthetic peptides. Culture medium of metabolically
labeled cells secreting PrP-sen(GPINEG) and other proteins
were precleared by centrifugation at 10,000 rpm for 10 min. The culture
supernatants were added to wells precoated with the designated
synthetic PrP peptides (200 µM, 50 µl/well) or PrP-res
(100 ng/40 µl/well). After incubation for 24 h, the cells were
washed four times and bound proteins were eluted with SDS-PAGE sample
buffer with heating. Half of each eluate volume was subjected to
SDS-PAGE and phosphor autoradiography. To confirm the identity PrP
bands in the bound fractions, we also used culture supernatants
depleted of PrP-sen by single round of immunoprecipitation with
anti-HaPrP219-232 rabbit serum and protein A-Sepharose (indicated by
D). The lane labeled
"PrP-sen(GPINEG)" shows
35S-PrP-sen(GPINEG) purified from metabolically
labeled culture supernatants by immunoprecipitation with the same
antiserum. Lanes labeled Culture sup. contain one-tenth
equivalent of the culture supernatants added to the binding reactions.
Arrowheads indicate three major bands of
35S-PrP-sen(GPINEG) bound to PrP synthetic
peptides or PrP-res. The lowest arrowhead marks an
N-terminal truncated fragment that is especially prominent in culture
supernatants as described previously (8).
-sheet aggregates. Thus, we analyzed the conformation of the
synthetic peptides described in this study to determine if they
exhibited a similar tendency to form
-sheet structure. The peptides
were dissolved in water and then diluted in conversion buffer in the
presence or absence of Sarkosyl to reproduce the manipulations used in
the cell-free conversion reactions and to evaluate the effect of
Sarkosyl on the conformation of the peptides. Circular dichroism (CD)
spectra of the Ha117-141, Ha121-141, Ha180-199, Ha200-223, and
Ha218-232 are shown in Fig. 8. The
spectra indicate that Sarkosyl has differential effects on the CD
spectra of the various peptides. The spectra of Ha117-141 and
Ha200-223 indicated changes in secondary structure from almost all
random coil, which is characterized by a negative ellipticity near 198 nm, to high
-sheet, characterized by strong positive ellipticity
near 198 nm and negative ellipticity near 220 nm (Fig. 8, b
and c). A similar, but much less pronounced effect of
Sarkosyl was seen with Ha180-199. The spectrum of Ha218-232 was
initially indicative of random coil and was unaffected by Sarkosyl. The
spectrum of Ha121-141 became more strongly indicative of random coil
in the presence of Sarkosyl. Interestingly, the secondary structure
changes in the peptides toward
-sheet by Sarkosyl correlated with
the relative abilities of the peptides to inhibit the conversion of
PrP-sen to PrP-res. The peptides with strong tendencies to form
increased
-sheet on addition of Sarkosyl (Ha117-141 and Ha200-223)
were the most efficient inhibitors of cell-free conversion of PrP-sen
(compare Fig. 8 to Fig. 2g). An intermediate effect was seen
with the weaker inhibitor Ha180-199 and no induction of
-sheet was
seen for the non-inhibitory peptides Ha121-141 and Ha218-232.
Ha121-141 was previously reported to form
-sheet secondary
structures (12) but that observation was made under different buffer
and solubilization conditions compared with the more physiologically
compatible conditions used in the present study.
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Fig. 8.
Circular dichroism spectra of synthetic PrP
peptides in the presence and absence of 0.1% Sarkosyl.
a, reference CD spectra of all- , all-
, and all-random
(Redrawn from: Ref. 42); b, Ha117-141; c,
Ha200-223; d, Ha180-199; e, Ha121-141;
f, Ha218-232. The final peptide concentrations were 1 mg/ml
peptide with or without 0.1% Sarkosyl in buffer containing 160 mM KCl, 4 mM MgCl2, 40 mM citrate (pH 6.0), 37 °C.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
10
µM required at least 60-fold stoichiometric excesses of
peptide over PrP molecules to exert their inhibitory effects. One
possible explanation for the need for stoichiometric excesses for
inhibition by these peptides is that formation of high
-sheet
aggregates or polymers of the PrP peptides may be required to compete
with PrP-res for binding to PrP-sen. This would be consistent with the
observations that peptides Ha109-141, Ha117-141, and Ha200-223 can
form
-sheet-rich structures according to Fourier transform infrared and/or CD analyses (see Ref. 12 and this study). The secondary structure of Ha166-179 could not be assessed with CD because
of the presence of the highly far UV-absorbent solvent dimethyl
sulfoxide, which was required for initial dissolution of this peptide.
However, a similar synthetic peptide of human PrP residues 169-185 was
reported to form an aggregate of rod-like structure and exhibit Congo
red birefringence (27), suggesting that Ha166-179 may form a
-sheet-rich structure. It is also possible that monomers of the PrP
peptides can bind to PrP-sen and inhibit conversion, but that
stoichiometric excess of inhibitory peptides is required because of the
relative affinities of the peptides and PrP-sen for PrP-res. Although
it remains to be elucidated which form of these inhibitory peptides
binds to PrP-sen, the binding itself suggests the peptides may serve as
useful probes of the sites of the PrP-sen-PrP-res interaction such as
the regions on the PrP-res molecule(s) which are involved in the interaction.
-strand and
second
-helix (29) plays an important role in PrP-res formation. By
contrast, a PrP mutant lacking residues 23-88 and 141-176 (PrP106) was able to form protease-resistant PrP (30, 31). In addition, an amber
mutation of human PrP at residue 145 caused
Gerstmann-Straussler-Scheinker syndrome with PrP plaques consisting of
mutant PrP (32). These findings suggest that the region corresponding
to Ha166-179 may not be essential for PrP-res formation. However, this
is apparently not generally true for PrP-res formation in TSE diseases
of infectious origin. For instance, it is well known that sheep
homozygous for Arg/Arg at residues 171 of PrP are resistant to scrapie
(reviewed in Ref. 33). In addition, substitutions of PrP residues 167 and 171 prevented PrP-res formation in scrapie-infected cell cultures (21). Taken together, these observations indicate that there are
multiple types of PrP-res formed and that the region corresponding to
residues 166-179 is involved in the formation of PrP-res from full-length PrP-sen precursors.
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ACKNOWLEDGEMENTS |
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We thank Gregory Raymond for technical assistance and Gary Hettrick and Anita Golden for graphics assistance.
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FOOTNOTES |
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* 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.
¶ Supported in part by a postdoctoral fellowship from the Natural Sciences and Engineering Research Council of Canada.
To whom correspondence should be addressed: Laboratory of
Persistent Viral Diseases, NIAID, National Institutes of Health, Rocky
Mountain Laboratories, 903 S. 4th St., Hamilton, MT 59840. Tel.:
406-363-9264; Fax: 406-363-9286; E-mail: bcaughey@nih.gov.
Published, JBC Papers in Press, February 1, 2001, DOI 10.1074/jbc.M100288200
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ABBREVIATIONS |
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The abbreviations used are: TSE, transmissible spongiform encephalopathy; PrP-res, proteinase-resistant prion protein; PrP-sen, proteinase-sensitive prion protein; PK, proteinase K; GPI, glycosylphosphatidylinositol; GPINEG, glycosylphosphatidylinositol-negative; GdnHCl, guanidine hydrochloride; CD, circular dichroism; PAGE, polyacrylamide gel electrophoresis.
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