From the Laboratory of Persistent Viral Diseases, NIAID, National Institutes of Health, Rocky Mountain Laboratories, Hamilton, Montana 59840
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ABSTRACT |
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The transmissible spongiform encephalopathies are
characterized by the conversion of the protease-sensitive prion protein (PrPsen) into a protease-resistant isoform (PrPres) associated with the
neuropathogenic process in vivo. Recently, PrPres has been
shown to be capable of directly inducing the conversion of PrPsen to
PrPres in a cell-free in vitro system. In the present experiments, various PrP peptides were studied for their ability to
enhance or inhibit this cell-free conversion reaction. None of the
synthetic peptides was able to confer protease-resistance to the
labeled PrPsen molecules on their own. On the contrary, peptides from
the central part of the hamster PrP sequence from 106 to 141 could
completely inhibit the conversion induced by preformed PrPres. The
presence of residues 119 and 120 from the highly hydrophobic sequence
AGAAAAGA (position 113 to 120) was crucial for an efficient inhibitory
effect. Fourier transform infrared spectroscopy analysis indicated that
inhibitory peptides formed high -sheet aggregates under the
conditions of the conversion reaction, but this was also true of
certain peptides that were not inhibitory. Thus, the potential to form
-sheeted aggregates may be necessary, but not sufficient, for
peptides to act as inhibitors of PrPres formation. Clearly, the amino
acid sequence of the peptide is also important for inhibition. The
sequence specificity of the inhibition is consistent with the idea that
residues in the vicinity of positions 106-141 of PrPres and/or PrPsen
are critically involved in the intermolecular interactions that lead to
PrPres formation.
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INTRODUCTION |
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Transmissible spongiform encephalopathies
(TSE)1 are fatal
neurodegenerative diseases including sporadic and familial
Creutzfeldt-Jakob disease, kuru and
Gerstmann-Sträussler-Scheinker syndrome in humans, scrapie in
sheep, and bovine spongiform encephalopathy in cattle (1, 2). TSE are
characterized by the formation and accumulation of an abnormal
proteinase K-resistant isoform (PrPres) of normal protease-sensitive
host-encoded prion protein (PrPsen). PrPres is formed from PrPsen by a
post-translational process involving conformational changes, a higher
-sheet content, and the formation of macromolecular aggregates
(3-5). PrPres is closely associated with TSE-mediated brain pathology,
suggesting a crucial role of PrPres in the pathogenic process. However,
in the absence of PrPsen expression in the brain tissue of recipients of scrapie-infected neurografts, no pathology outside the graft itself
was observed, demonstrating that PrPres and PrPsen are both required
for the pathology (6). Both cross-species transmission in
vivo and efficient PrPres accumulation in vitro require
a high degree of sequence homology between PrPres and PrPsen molecules. For example, in scrapie-infected neuroblastoma cells, point mutations at three positions, 108, 111, and 138, in the mouse PrP sequence are
sufficient to completely block the formation of PrPres (7, 8). In a
cell-free in vitro system, PrPres induces the conversion of
PrPsen to PrPres (9). This interaction between PrPres and PrPsen occurs
with species and strain specificities that mimic TSE species barrier
effects and strain differences in vivo (10-13). Collectively, these in vivo and in vitro
observations strongly suggest that precise direct interactions between
PrPres and PrPsen are critical in PrPres formation and TSE
pathogenesis.
In previous experiments, small synthetic peptides containing certain
PrP sequences spontaneously aggregated to form fibrils with a high
degree of -sheet secondary structure (14-17). Moreover, other
synthetic PrP peptides were shown to interact with PrPsen molecules,
forming an aggregated complex with increased protease resistance (18,
19). In the current study, peptides from a variety of locations in the
hamster PrP sequence were studied for their ability to influence the
generation of PrPres under cell-free conditions. A group of peptides
from the central portion of the PrP molecule having high
-sheet
content displayed a strong inhibitory effect on the cell-free
conversion assay. Based on these results, we propose the existence of
sites on the central part of the PrP molecule that allow a specific
binding between PrPsen and PrPres as a first step in the conversion of
PrPsen to PrPres.
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EXPERIMENTAL PROCEDURES |
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Peptides--
The following peptides were synthesized by the
laboratory of molecular structure of NIAID, NIH, Rockville MD: hamster
P106-128 (KTNMKHMAGAAAAGAVVGGLGGY), P109-141
(MKHMAGAAAAGAVVGGLGGYMLGSAMSRPMMHF), P113-141
(AGAAAAGAVVGGLGGYMLGSAMSRPMMHF), and P121-141
(VVGGLGGYMLGSAMSRPMMHF). Peptides were >95% pure, and analysis
by high pressure liquid chromatography revealed only a single peak.
Alzheimer's disease amyloid- protein fragment 1-40 (A
-(1-40))
was purchased from Sigma. Lyophilized peptides were dissolved in
deionized water at a concentration of 2 mM, distributed
into 20-µl aliquots, and stored at
20 °C.
Labeling and Purification of PrPsen-- The radiolabeling and the purification of the 35S-PrPsen were performed as described previously by Raymond et al. (13). Briefly, 90% confluent cells were cultured for 30 min at 37 °C in 1.5 ml of methionine/cysteine-deficient medium followed by a 90-120-min incubation with 1.4 mCi of [35S]methionine/cysteine/25-cm2 flask. Then the cells were washed twice with phosphate-buffered saline (20 mM sodium phosphate, pH 7.4, 130 mM NaCl) and lysed in 1.5 ml of lysis buffer containing 5 mM Tris-HCl, pH 7.4, 140 mM NaCl, 5 mM EDTA, 0.5% sodium deoxycholate, and 0.5% Triton X-100. The proteins were precipitated by the addition of 5 volumes of methanol, resuspended in detergent-lipid complexes, and immunoprecipitated by overnight incubation at 4 °C with anti-PrP 3F4 monoclonal antibody (20). Immune complexes were collected by binding to protein A-Sepharose beads. Radiolabeled PrPsen was eluted from Sepharose beads using 0.1 M acetic acid and stored at 4 °C until use. The 35S-labeled hamster PrPsen used in this study was the glycophosphatidylinositol-negative form (35S-HaPrPsen) described previously (9).
Purification and Analysis of PrPres--
PrPres was purified
from brains of scrapie-infected Syrian golden hamsters by detergent
lysis and differential centrifugation (21). Hamsters were infected 70 days before with the hamster scrapie strain 263K. The yield of PrPres
was determined by Western blotting using a rabbit polyclonal PrP
antiserum (R27) raised against a synthetic hamster PrP peptide
(residues 89-103) (22). The purity of the preparations was estimated
at 50-60% by silver-staining of SDS-PAGE gels. The hamster PrPres
preparation (HaPrPres) was then diluted to 1 mg/ml in
phosphate-buffered saline containing 1% sulfobetaine 3-14 and stored
at 20 °C.
Cell-free Conversion Assay--
The cell-free conversion
reaction was performed as described previously (9, 13). Briefly,
purified PrPres was partially denatured with 2.5 M
guanidine hydrochloride for 30-60 min at 37 °C. An aliquot of 200 ng of HaPrPres, typically 8 µl, was then incubated for 40 h at
37 °C with ~1 ng of immunopurified 35S-PrPsen
(~12,000 cpm/reaction) in a final volume of 20 µl of conversion
buffer (50 mM sodium citrate, pH 6, 5 mM
cetylpyridinium chloride, 0.625% N-lauryl sarcosinate) in
the presence or absence of peptides. At the end of the incubation time,
each reaction was split 1:10, the major fraction was digested with 100 µg/ml proteinase K (PK) in Tris-saline buffer (50 mM
Tris, pH 8, 130 mM NaCl) for 1 h at 37 °C, and the
minor part (PK) was reserved as an undigested control. The PK
reaction was stopped by the addition of 10 µl of a mixture of 4 mg/ml
thyroglobulin, 20 mM Pefabloc (Boehringer-Mannheim) to each
fraction (+ and
PK). Samples were then precipitated in 5 volumes of
methanol and centrifuged for 20 min at 14,000 × g. The
pellets were resuspended in sample buffer (65 mM Tris-HCl,
pH 6.8, 5% glycerol, 5% SDS, 4 M urea, 5%
-mercaptoethanol, 0.5% bromphenol blue), boiled 5 min, and analyzed
by SDS-PAGE on NOVEX pre-cast gels. The percent of the conversion was
calculated by the ratio between the PK-resistant
35S-labeled bands of approximate molecular masses 28-30
kDa and the nondigested 35S-PrPsen as quantified by
phosphor autoradiographic imager analysis. At the concentration used,
none of the peptides affected the PK digestion of PrPres (data not
shown).
Sedimentation Experiments--
Immunopurified
35S-HaPrPsen (12,000 cpm/reaction) was incubated for
24 h at 37 °C with 200 ng of 2.5 M guanidine
hydrochloride-pretreated HaPrPres in the absence or presence of
indicated concentrations of peptides P-(109-141), P-(113-141),
P-(121-141), and A-(1-40). At the end of the incubation time, the
samples were centrifuged at room temperature for 20 min at 14,000 × g. The supernatants and the pellets were separately
collected and methanol-precipitated before SDS-PAGE analysis and
phosphor autoradiographic imager quantification.
Fourier Transform Infrared Spectroscopy--
The dried peptides
were rehydrated by mixing with conversion buffer and incubated for
4-60 h at room temperature before Fourier transform infrared
spectroscopy analysis. Samples (6 µl) were loaded into variable
path-length cells with CaF2 plates adjusted to a path
length of 6 µm. After purging the sample chamber to reduce the water
vapor contribution, spectra were collected with a Perkin-Elmer system
2000 spectrometer. Spectral parameters were as follows: 254 scans;
4-cm1 resolution; 1 cm/sec optical path difference
velocity; Kaiser-Bessel apodization. Buffer spectra were subtracted
from peptide spectra to give a flat base line in the
1800-2200-cm
1 region. A water vapor spectrum was then
subtracted to minimize the water vapor bands in the
1750-1850-cm
1 region.
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RESULTS |
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Inhibition of Conversion by Hamster PrP Peptides--
Various PrP
synthetic peptides, whose localization on the primary hamster PrP
sequence is shown in Fig. 1A,
were tested for effects on the in vitro conversion of
metabolically labeled PrPsen molecules into PrPres. The peptides were
used in the cell-free conversion assay at a final concentration of 0.8 mM. As shown in Fig. 1B, the non-PK-treated
35S-HaPrPsen appeared as a double band with molecular
masses of 30 and 28.5 kDa, corresponding to mono- or unglycosylated
forms of the molecule, respectively. In the absence of HaPrPres, no PK-resistant bands were seen in the control experiment. In those conditions, none of the peptides was able by itself to promote the
conversion of 35S-HaPrPsen to a PK-resistant form (Fig.
1B, lanes marked ). In the presence of 200 ng
of HaPrPres, two PK-resistant 35S-labeled bands were
obtained with molecular masses of 24 and 20 kDa (Fig. 1B,
lanes marked +). However, two synthetic hamster peptides,
P-(106-128) and P-(109-141), were able to inhibit the formation of
35S-labeled PK-resistant PrP bands. A third peptide,
P-(89-103), partially inhibited the conversion, and six other
peptides, P-(23-37), P-(57-64), P-(57-72), P-(57-80), P-(139-170),
and P-(218-232) failed to inhibit the conversion.
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Importance of the 8-Amino Acid Sequence 113-120-- The PrP synthetic peptide AGAAAAGA from position 113 to 120 has been described as the most highly amyloidogenic peptide in the protein (14). To assess the influence of the AGAAAAGA sequence contained in both P-(106-128) and P-(109-141), we synthesized two peptides differing only in this hydrophobic sequence, P-(113-141) and P-(121-141). The P-(113-141) peptide sequence efficiently inhibited the in vitro conversion with an IC50 value of 40 µM (Figs. 3, A and B). In contrast, the P-(121-141) peptide failed to inhibit the conversion at all of the concentrations used (Figs. 3, A and B). When incubated with the labeled PrPsen in the absence of PrPres, none of these peptides conferred PK resistance to PrPsen molecules (Fig. 3A). Thus, the hydrophobic sequence from 113 to 120 appeared to be essential to the inhibitory effect of peptides from this region of PrP.
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Conformational Analysis of Inhibitory Versus Noninhibitory
Peptides--
Fourier transform infrared spectroscopy was used to
compare the conformations of the PrP and A-(1-40) peptides to see
if there was a correlation between inhibitory efficacy and
conformation. For this purpose, peptides were incubated for 4-60 h in
conversion buffer without any PrPsen or PrPres. Fourier transform
infrared spectra of peptides incubated for 24 h at a concentration
of 2 mM are shown in Fig. 4.
Five of the peptides (P-(106-128), P-(109-141), P-(113-141),
P-(121-141), and A
-(1-40)) showed prominent absorbance maxima at
~1630 cm
1 that were indicative of a high
-sheet
content. For P-(109-141), this predominant absorbance at 1630 cm
1 was maintained down to concentrations of 0.2 mM. These same five peptides were at least partially
insoluble as each formed a visible particulate suspension. Two other
PrP peptides (P-(89-103) and P-(218-232)) maintained clear solutions
and had absorbance maxima near 1667 cm
1, with no evidence
of the
-sheet absorbance at ~1630 cm
1. Although all
the inhibitory peptides showed evidence of a high
-sheet content,
other peptides, such as P-(121-141) and A
-(1-40), showed a similar
-sheet content but gave no inhibition of conversion. Thus, the
potential to form a high amount of
-sheet structure might be
necessary, but not sufficient, for the specific inhibitory activity of
any given peptide.
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Peptide Sedimentation Studies--
Considering the propensity of
hydrophobic PrP peptides to aggregate in vitro, we
investigated whether the inhibition of the conversion could be
explained by peptide-induced aggregation of 35S-HaPrPsen,
which might prevent conversion of this precursor protein. For this
purpose, conversion reaction mixtures were centrifuged in the presence
or absence of various peptides, and pellet and supernatant fractions
were analyzed for 35S-HaPrPsen (Fig.
5). Whereas 35S-HaPrPsen
remained soluble in the absence of peptides, incubation with either
inhibitory peptides (P-(109-141), P-(113-141)) or noninhibitory
peptides (P-(121-141), A-(1-40)) caused aggregation and pelleting
of 35S-HaPrPsen. Thus, although peptide-induced aggregation
of PrPsen occurred, this sedimentation was not sufficient for the
inhibition of the conversion reaction.
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DISCUSSION |
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In the present report, we used PrP synthetic peptides to map portions of the molecule that may be involved in PrPres-PrPsen interactions. Peptides from the central part of PrP sequence, P-(106-128), P-(109-141), and P-(113-141), were very efficient in inhibiting the in vitro cell-free conversion (30 µM < IC50 < 230 µM). The inhibition of the cell-free conversion assay induced by such peptides suggests that specific interactions between PrPsen and PrPres may involve the central region of the molecule. Recently, Hölscher et al. (23) showed that a mutant mouse PrP lacking the sequence from 114 to 121 (AGAAAAGA) is not converted into a proteinase K-resistant isoform after expression in scrapie-infected mouse neuroblastoma cells. In our current studies, the presence of the corresponding hamster sequence from 113 to 120 correlated with the inhibition of the conversion reaction. Indeed, all the peptides having this sequence such as P-(106-128), P-(109-1410, and P-(113-141) had an inhibitory effect, whereas P-(121-141) did not exhibit this property (Figs. 3, A and B). Interestingly, residues 119 and 120 appeared to be most critical to the inhibition as P-(119-141) was nearly as effective as P-(113-141) (Fig. 3B). Peptides from the amino-terminal region such as P-(23-37), P-(57-64), P-(57-72), and P-(57-80) and from the carboxyl-terminal region such as P-(139-170) and P-(218-232) did not inhibit the conversion reaction. Only P-(89-103) was able to slightly decrease the conversion when incubated at the high concentration of 0.8 mM (Fig. 1B). P-(89-103) is contained in the PK-resistant core of PrPres, and its location is adjacent to the most inhibitory peptide sequences. These data suggested that, unlike the central portion of PrPres, the amino- and the carboxyl-terminal regions of the molecule are less involved in the intermolecular interactions resulting in PrPres formation. This explanation is consistent with the fact that the carboxyl- and amino-terminal portions of PrPres are more sensitive to the PK digestion than the central part (21, 24).
In our experiments, the presence of PrP amino acid residues from 113 to 120 were required for maximal inhibition, but other amino acid residues are likely also to be involved in the specific intermolecular interaction. The adjacent sequence from residues 129 to 141 seemed to play an important modulatory role in the inhibition of conversion, since P-(106-128) was significantly less inhibitory compared with P-(109-141) (Figs. 2, A and B). This interpretation is consistent with a previous report showing that a point mutation at position 138 could block the accumulation of PrPres in the mouse neuroblastoma cell system (8). Together these results suggest that the region of residue 138 in the PrP polypeptide is important in the intermolecular interactions leading to PrPres formation.
The mechanism of action of the inhibitory peptides is still unclear.
Possibly the inhibitory peptides mimic the structure of PrPres and
competitively bind to PrPsen molecules to block the conversion process.
Alternatively, peptides could bind to PrPres itself, blocking further
interaction with PrPsen. In the conversion reaction buffer, the
peptides P-(106-128), P-(109-141), and P-(113-141) possessed a high
-sheet content reminiscent to brain-derived PrPres (3-5). Whether
the active state of the inhibitory peptides is the soluble or
aggregated form is not known. However, their mechanism of action cannot
be explained merely by their tendency to form
-sheet structure and
sedimentable aggregates with PrPsen, since noninhibitory peptides such
as P-(121-141) and A
-(1-40) displayed the same biochemical
properties.
In our hands, none of the tested peptides were capable of conferring PK resistance to 35S-PrPsen (Fig. 1B). Using slightly different conditions, other authors have shown that incubation of hamster PrPsen with hamster peptide P-(90-145) resulted in the formation of a PK-resistant, sedimentable PrPsen·peptide complex (18, 19). Upon PK digestion, no shift of the molecular mass of this PrPsen·peptide complex was reported. Thus, this complex may be a large aggregate unaccessible to PK, in contrast to brain-derived PrPres and PrPres formed in our cell-free conversion experiments, which loses ~67 amino-terminal amino acid residues (6-7 kDa) with PK treatment.
Studies using PrP may suggest new approaches for possible therapeutic intervention. The ability of PrP peptides to inhibit the accumulation of PrPres in vivo remains to be established. Nevertheless, PrP peptides dispensed by direct injection or delivered by gene therapy might provide specific therapeutic treatment for TSE diseases. Furthermore, structural analysis of the minimal peptide sequence required for inhibition of conversion might lead to development of nonpeptide compounds with therapeutic potential.
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ACKNOWLEDGEMENTS |
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We wish to thank Gary Hettrick for help with the preparation of the figures, Dr. Jan Lukszo for synthesis and purification of peptides, Dr. Suzette A. Priola for stimulating discussions, and Drs. Heidi Super, Jim Fox, and Kim Hasenkrug for critical reading of the manuscript.
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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 by INSERM and by the Fondation pour la Recherche
Médicale.
§ To whom correspondence should be addressed: Laboratory of Persistent Viral Diseases, NIAID, National Institutes of Health, Rocky Mountain Laboratories, 903 South 4th St., Hamilton, MT 59840. Tel.: 406-363-9354; Fax: 406-363-9204; E-mail: bchesebro{at}nih.gov.
1
The abbreviations used are: TSE, transmissible
spongiform encephalopathies; PrP, prion protein; PrPsen,
protease-sensitive prion protein; PrPres, protease-resistant prion
protein; A-(1-40), amyloid-
protein fragment 1-40; HaPrPsen,
hamster PrPsen; PAGE, polyacrylamide gel electrophoresis; PK,
proteinase K.
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REFERENCES |
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