Inhibition of Interactions and Interconversions of Prion Protein Isoforms by Peptide Fragments from the C-terminal Folded Domain*

Motohiro HoriuchiDagger §, Gerald S. BaronDagger , Liang-Wen XiongDagger , and Byron CaugheyDagger ||

From the Dagger  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

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 beta -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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 -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 beta -strands (beta 1 and beta 2) and hatched boxes indicate regions forming alpha -helix (alpha 1, alpha 2, and alpha 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.

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.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.


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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.

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.


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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.

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.


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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 alpha 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.

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.


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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.

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.


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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).

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.


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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).

Secondary Structures of the Inhibitory Peptides-- Inhibitory peptides described by Chabry et al. (12) tended to form high beta -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 beta -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 beta -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 beta -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 beta -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 beta -sheet was seen for the non-inhibitory peptides Ha121-141 and Ha218-232. Ha121-141 was previously reported to form beta -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-alpha , all-beta , 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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 >= 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 beta -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 beta -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 beta -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.

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 beta -strand and second alpha -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.

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.

    ACKNOWLEDGEMENTS

We thank Gregory Raymond for technical assistance and Gary Hettrick and Anita Golden for graphics assistance.

    FOOTNOTES

* 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

    ABBREVIATIONS

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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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