jbc papers in press. published on february 1, 2001 as manuscript

1/26th/01 h:00 am/pm.

Inhibition of interactions and interconversions of prion protein isoforms by peptide fragments

from the C-terminal folded domain

Motohiro Horiuchi1,2, Gerald S. Baron1, Liang-Wen Xiong1 and Byron Caughey1

1Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, NIAID, NIH, Hamilton,

Montana 59840 USA

2Department of Veterinary Public Health and the National Research Center for Protozoan

Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro Hokkaido 080-

8555 Japan

Running title: PrP peptide inhibitors of PrP-res formation

Correspondence to:Dr. Byron Caughey,Laboratory of Persistent Viral Diseases,NIAID, NIH, Rocky Mountain Laboratories,903 S 4th St.,Hamilton, MT 59840 USAPhone: 406-363-9264Fax: 406-363-9286e-mail: [email protected]

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Summary

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.

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Introduction

Transmissible spongiform encephalopathies (TSEs) 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 preeminent neuropathological feature of TSEs is the accumulation of

the disease-specific, protease-resistant isoform of prion protein, designated as PrP-res or

PrPSc, in 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

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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

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-

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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-

223 were first dissolved in dimethylsulfoxide (DMSO) to make 10 mM stock solutions. Aliquots

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of each stock solution were stored at –20°C until use.

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 sec, 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 of 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 (Boehringer Mannheim) to 2 mM,

thyroglobulin to 20 µg/ml as a carrier protein, and four 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 multi-well 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 PBS and then blocked with 0.5% skim milk in PBS at room temperature for 2 h. After

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washing the wells once with PBS, 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 1x sample buffer (5% SDS, 4M urea, 62.5 mM Tris-HCl (pH

6.8), 3 mM EDTA, 5% glycerol, 5% 2-mercaptoethanol, 0.04% bromophenol 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 crosslinked 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 PBS 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

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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 4M

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), 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 bandwidth, dual beam mode, 400 kHz sampling rate, 2 V high volts criterion. Wavelength

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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 90 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

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 guanidine hydrochloride (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

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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 ~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 to 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. 2b,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. 2c,e,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. 2d,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

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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 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 non-specific 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 lane). 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

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serum (NRS). 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 multi-well 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

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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

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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 β-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 β-sheet structure. The

peptides were dissolved in water and then diluted in conversion buffer in the presence or

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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 β-sheet, characterized by strong positive ellipticity

near 198 nm and negative ellipticity near 220 nm (Fig. 8b,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 towards β-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 β-sheet on addition of sarkosyl (Ha117-

141 and Ha 200-223) 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 β-sheet was seen for the non-inhibitory peptides Ha121-141 and

Ha218-232. Ha121-141 was previously reported to form β-sheet secondary structures (12)

but that observation was made at under different buffer and solubilization conditions

compared to the more physiologically compatible conditions used in the present study.

Discussion

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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 (IC50s) 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

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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 over-stabilize PrP-sen or causes 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

IC50s 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 β-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 Ha 200-223 can form β-sheet rich structures according to FT-IR and/or CD

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analyses [(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 DMSO, which was

required for initial dissolution of this peptide. However, a similar synthetic peptide of human PrP

residues169-185 was reported to form an aggregate of rod-like structure and exhibit Congo red

birefringence (27), suggesting that Ha166-179 may form β-sheet rich structure. It is also

possible that monomers of the PrP peptides can bind to PrP-sen and inhibit conversion, but that

stoichiometric excesses 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

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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 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 µM 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 a loop structure

between the second β-strand and second α-helix (29) plays an important role in PrP-res

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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 (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 multi-well plates may provide high throughput screens for

compounds that inhibit those interactions.

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Acknowledgements

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

for graphics assistance. G.S.B. was supported in part by a post-doctoral fellowship from the

Natural Sciences and Engineering Research Council of Canada.

FOOTNOTES

a Abbreviations: TSE, transmissible spongiform encephalopathy; PrP-res, proteinase-resistant

prion protein; PrP-sen, proteinase-sensitive prion protein; PK, proteinase K; PBS, phosphate

buffered saline; GPI, glycosylphosphatidylinositol; GPINEG, glycosylphosphatidylinositol-

negative; GdnHCl, guanidine hydrochloride. CD, circular dichroism.

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Figure legends

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 β-

strands (β1 and β2) and hatched boxes indicate regions forming α-helix (α1, α2, and α3)

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.

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 +/- SD (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.

Fig. 4. Solid phase binding and conversion reactions. 35S-PrP-sen(GPINEG) was incubated

for 2 d 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 α219-232, 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 Figure 2.

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 Figure 4, peptides were added in

increasing concentrations to the binding buffer with 35S-PrP-sen(GPINEG). After incubation

for 2 d, the wells were washed and assayed for bound 35S-GPI(-)PrP. The data points indicate

means +/- SD (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.

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

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described in Experimental Procedures. Supernatants were saved as the unbound fraction and

the 35S-GPI(-)PrP eluted from the wells with 5% SDS and 4 M Urea by heating at 80°C was

designated the bound fraction. The graph shows means +/- SD (n=3) of the percentage of the

bound 35S-GPI(-)PrP against the total 35S-GPI(-)PrP in the reaction (the sum of the unbound

and bound fractions).

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 pre-

coated with the designated synthetic PrP peptides (200 µM, 50 µl/well) or PrP-res (100 ng/40

µl/well). After incubation for 24 hr, 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 a major three bands of 35S-PrP-sen(GPINEG)

bound to PrP synthetic peptides or PrP-res. The lowest arrowhead marks an N-terminally

truncated fragment that is especially prominent in culture supernatants as described previously

(8).

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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:

Brahms, s., Brahms, J., J. Mol. Biol., 1980, 138: 149-178); (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.

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PrP

-sen

(GP

INE

G)

500 50 5 0

500

+PK

PrP

-sen

(GP

INE

G)

500 50 5 0

500

PrP

-sen

(GP

INE

G)

500 50 5 0

500

+PK +PK+ + + + - + + + + - + + + + -

a b c

d e f

28.3

20.5

28.3

20.5

(mM

)

:PrP-res

Fig. 2a-f. Horiuchi et al.


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50050 5 0

500

PrP

-sen

(GP

INE

G)

+PK

28.5

20.8

+ -+++

50050 5 0

500

PrP

-sen

(GP

INE

G)

+PK

+ -+++ :PrP-res

(mM

)

a b

Fig. 3. Horiuchi et al.


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+- ++++

+ + +- ----

- -PrP-res:

PK:

a219-232 NRSAbs:

28.3

20.8

Fig. 4. Horiuchi et al.


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DD

DD

DD

DD

D

Culture sup.

None

aa109-141

aa121-141

aa166-179

aa180-199

aa200-223

aa218-223

PrP-res

PrP-sen(GPINEG)

Bound to P

rP synthetic peptide or P

rP-res

39.8

30.0

16.6

Fig. 7. H

oriuchi et al.

by guest on February 3, 2018 http://www.jbc.org/ Downloaded from

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Motohiro Horiuchi, Gerald S. Baron, Liang-Wen Xiong and Byron Caugheyfragments from the C-terminal folded domain

Inhibition of interactions and interconversions of prion protein isoforms by peptide

published online February 1, 2001J. Biol. Chem.

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