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Published as doi: 10.1096/fj.07-099549.
 (The FASEB Journal. 2008;22:2177-2184.)
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Antiprion properties of prion protein-derived cell-penetrating peptides

Kajsa Löfgren*,1, Anna Wahlström*, Pontus Lundberg{dagger}, Ülo Langel{dagger}, Astrid Gräslund*,1 and Katarina Bedecs*

* Department of Biochemistry and Biophysics, The Arrhenius Laboratories; and

{dagger} Department of Neurochemistry and Neurotoxicology, Stockholm University, Stockholm, Sweden

1Correspondence: Department of Biochemistry and Biophysics, The Arrhenius Laboratories, Stockholm University, SE-106 91 Stockholm, Sweden. E-mail: K.L., lofgren{at}dbb.su.se; A.G., astrid{at}dbb.su.se


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In prion diseases, the cellular prion protein (PrPC) becomes misfolded into the pathogenic scrapie isoform (PrPSc) responsible for prion infectivity. We show here that peptides derived from the prion protein N terminus have potent antiprion effects. These peptides are composed of a hydrophobic sequence followed by a basic segment. They are known to have cell-penetrating ability like regular cell-penetrating peptides (CPPs), short peptides that can penetrate cellular membranes. Healthy (GT1–1) and scrapie-infected (ScGT1–1) mouse neuronal hypothalamic cells were treated with various CPPs, including the prion protein-derived CPPs. Lysates were analyzed for altered protein levels of PrPC or PrPSc. Treatment with the prion protein-derived CPPs mouse mPrP1–28 or bovine bPrP1–30 significantly reduced PrPSc levels in prion-infected cells but had no effect on PrPC levels in noninfected cells. Further, presence of prion protein-derived CPPs significantly prolonged the time before infection was manifested when infecting GT1–1 cells with scrapie. Treatment with other CPPs (penetratin, transportan-10, or poly-L-arginine) or prion protein-derived peptides lacking CPP function (mPrP23–28, mPrP19–30, or mPrP23–50) had no effect on PrPSc levels. The results suggest a mechanism by which the signal sequence guides the prion protein-derived CPP into a cellular compartment, where the basic segment binds specifically to PrPSc and disables formation of prions.—Löfgren, K., Wahlström, A., Lundberg, P., Langel, U., Gräslund, A., and Bedecs, K. Antiprion properties of prion protein-derived cell-penetrating peptides.


Key Words: scrapie • prion conversion • N terminus • therapy • signal peptide


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
SPONGIFORM ENCEPHALOPATHIES, OR prion diseases, are fatal and incurable neurodegenerative conditions that occur in humans and other mammals. As a disease hallmark, the endogenous cellular prion protein (PrPC) becomes misfolded into a disease-related isoform called scrapie (PrPSc). The conformation of PrPSc makes it largely resistant to cellular degradation, and PrPSc accumulation in the brain is considered to cause neurodegeneration and death (1) .

PrPC is constitutively expressed in the adult brain, and the mature protein consists of amino acids 23–230 in human PrP. In aqueous solution, residues 126–228 constitute a regularly folded protein domain, whereas residues 23–125 constitute an unfolded, highly dynamic domain (2) . Many functional aspects of PrPC are associated with the N-terminal part. Residues 1–22 constitute a very hydrophobic signal sequence promoting entry into the endoplasmic reticulum (ER) and are normally cleaved off before PrPC reaches the cell surface (3) . Residues 23–30 form a basic segment and constitute one of two independent nuclear localization signal-like (NLS-like) segments of PrPC, although the functions of these NLS-like sequences remain largely unclear (4) . This NLS-like sequence is also called the preoctarepeat region, since it precedes five 8-aa sequence repeats, the so-called octarepeats. The octarepeats contain Cu2+ ion-binding sites, corresponding to a probable function of PrPC in cellular copper uptake (5) . Further, the preoctarepeat and octarepeat region (aa 23–100) is responsible for endocytotic internalization of PrPC. A glycosylphosphatidylinositol (GPI) anchor in the C terminus localizes PrPC to functionally specialized membrane domains, so-called lipid rafts (6 , 7) . Interestingly, a PrPC N-terminal region may also contribute in localization of PrPC to lipid rafts (8) .

The localization of PrPC to lipid rafts is suggested to be critical for its conversion into PrPSc and for prion infection (8) . The gradually lowered pH in the endocytic pathway of the recycling PrPC should be important for the prion conversion event (9 , 10) . Further, several cellular components have been proposed to be necessary for conversion to occur. Cell surface heparan sulfate (HS) carbohydrate chains seem to function as important conversion mediators (11 12 13) . HS is found attached to heparan sulfate proteoglycans (HSPGs), which presumably play a role in PrPC internalization (14 , 15) .

The hydrophobic PrP signal peptide is relatively insoluble in water. However, if combined with the positively charged NLS-like/preoctarepeat sequence (residues 23–38 in the mouse PrP sequence), the resulting peptide will become water soluble. The corresponding sequence is an amphipathic cell-penetrating peptide (CPP) (16 , 17) . CPPs are heterogeneous groups of short peptides capable of penetrating cellular membranes and translocating linked macromolecules into a variety of cells. Many different CPP internalization mechanisms have been proposed. A CPP may exhibit different entry mechanisms with or without attached cargo or in different model systems (18) . Association with membrane phospholipids and/or HSPGs presumably initiates CPP translocation (19 20 21 22) . CPPs include helical peptides of an amphipathic nature, such as transportan, as well as cationic arginine-rich peptides, such as penetratin (18) .

The prion protein-derived CPPs (PrP-CPPs) consist of the N-terminal signal peptide comprising residues 1–22 in mouse (m) PrP and 1–24 in bovine (b) PrP, coupled to sequences 23–28 and 25–30, respectively. The PrP-CPPs have been found to transport hydrophilic cargos across cell membranes (16 , 23) , in a process described as a lipid raft-dependent uptake by macropinocytosis (24) . When fluorescein-coupled mPrP1–28 was added to mouse neuroblastoma N2a cells, the peptide was found in endosomes or in a perinuclear pattern coinciding with the Golgi apparatus and as a diffuse cytoplasmic localization (16) . Likewise, fluoresceinyl-labeled bPrP1–30 translocated in a similar pattern into CHO cells (17) . Membrane perturbation studies in a lipid vesicle model system suggest that the PrP-CPPs cause transient pore formation (24) . Macropinocytosed CPPs may escape endosomes and avoid lysosomal degradation, since a transmembrane pH gradient imitating the one arising in endosomes in vivo was shown to promote escape of bPrP1–30 from lipid vesicles (23) . However, if the peptides are attached to HS or another cargo, like fluorescein, the fraction escaping the vesicles is considerably lower (23) .

The present study aimed to investigate whether treatment with various CPPs could antagonize conversion of PrPC to PrPSc. The unexpected results show that PrP-CPPs significantly lower the PrPSc levels in scrapie-infected mouse hypothalamic cell lines (ScGT1–1). This effect is specific for the PrP-CPPs among the CPPs and PrP peptides investigated. Further, no significant effects on the PrPC levels in healthy cells could be detected after PrP-CPP treatments, potentially coinciding with a specific interaction of the PrP-CPPs with PrPSc.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
GT1–1 cells (25) were a kind gift from Stanley B. Prusiner (University of California, San Francisco, CA, USA) All cell culture reagents and culture plates were purchased from Invitrogen AB (Lidingö, Sweden). Peptides penetratin, transportan-10 (TP-10), mPrP1–28, and 16-mer arginine (R16) were synthesized as described elsewhere (26) . Peptides mPrP1–28, mPrP1–28R, mPrP23–28, mPrP19–30, mPrP23–50, and bPrP1–30 were purchased from Neosystem Laboratoire (Strasbourg, France). Scrambled mPrP1–28R (randomized) peptide was designed using the Bio-Web DNA/Protein Sequence Randomizer (Web-based open-source Python CGI Script; http://www.cellbiol.com). All peptide sequences including terminal modifications are enlisted in Table 1 . Primary goat anti-mouse PrP (M-20) sc-1694 antibody and secondary donkey anti-goat peroxidase-conjugated antibody were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All other reagents were from Sigma-Aldrich Sweden AB (Stockholm, Sweden).


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  		Table 1. Peptides used for treatment of GT1–1 and ScGT1–1 cells

Cell cultures and RML infection
GT1–1 cells are murine neuronal hypothalamic cells. Brain homogenate from Rocky Mountain Laboratories (RML) prion-infected CD-1 mice was kindly provided by S. B. Prusiner. GT1–1 cells were infected with RML brain homogenate as described previously, at two separate occasions, generating chronically prion-infected cell lines (ScGT1–1a and ScGT1–1b) (27 28 29) . Cells were cultured as described previously (28) . ScGT1–1a/b cell lines were regularly tested for PrPSc infection and exhibited proteinase K (PK) resistance. ScGT1–1a/b cells can be cured from PrPSc propagation by pentosan polysulfate (PPS) treatment.

Peptide treatments
Peptide treatments of cell cultures
GT1–1, ScGT1–1a, or ScGT1–1b cells (0.2x106/well) were seeded out in 12-well Petri plates 9 days before harvest and analysis of PrP levels. The peptides, as listed in Table 1 , were tested for effects on PrPC levels in GT1–1 cells and effects on PrPSc levels in ScGT1–1 cells. In general, peptide treatments were made at final concentrations of 0.1, 0.5, 1.0, and 2.0 µM or as indicated, over 3, 5, or 8 days. Additions of phosphate-buffered saline (PBS) were used for negative controls. For positive controls, cells were treated with 2 µM of PPS. During 8 days of treatments with peptides at concentrations of 5 or 10 µM, the medium was changed every 24 h prior to new peptide addition. This was done in order to avoid toxicity from aggregated peptide not taken up into the cells. ScGT1–1b cells were cultured across several passages (8, 15, 23, and 30 days) in the presence of 0.5 µM mPrP1–28. For all treatments, new peptide was added every 24 h.

Peptide treatments during RML infection of GT1–1 cells
GT1–1 cells (0.2x106/well) were seeded out in 12-well Petri plates and cultured to reach ~80% confluence. GT1–1 cells were incubated with 0.05% RML brain homogenate as described (27 28 29) in the presence of 10 µM of either mPrP1–28, mPrP23–50, bPrP1–30, penetratin, TP-10, or 50 µM of mPrP23–28. New peptide additions were made every 24 h during the 3 days of RML infection. Control cells were infected with RML with addition of PBS instead of peptide. After RML and peptide treatments were terminated, the cultures were passaged for 3 wk before subsets of each culture were harvested and analyzed for presence of PrPSc by PK and Western blot. Remaining subsets were cultured for an additional 2 wk before harvest and analysis.

Peptide effect in binding study using precipitation of prion protein with heparin-conjugated agarose
GT1–1 or ScGT1–1b cells (2x106) were seeded out in 10 cm Petri dishes and cultivated for 7 days. Cells were extracted in cold lysis buffer (0.5% Triton X-100; 0.5% NaDoc; 150 mM NaCl; 10 mM EDTA; 50 mM Tris, pH 7.5, at 0°C) supplemented with protease inhibitors [10 mM phenyl-methanesulphonylfluoride (PMSF), 1 µg/ml pepstatin, 1 mg/ml aprotinin, and 1 mM sodiumorthovanadate]. The lysates were cleared and the protein levels were normalized as described below. Protein (500 µg) was used for each sample. Heparin-agarose beads (50 µl) were preincubated for 3 h at room temperature with 200 µM of mPrP1–28 or with lysis buffer as control. In parallel, lysates were preincubated with 100 µM mPrP1–28 or with lysis buffer as control. To preincubated heparin-agarose, lysate was added. To preincubated lysate, 50 µl of heparin-agarose beads was added. The final volume of all samples was 300 µl, and for samples with mPrP1–28, the final concentration of peptide was 67 µM. The samples were incubated for 3 h at room temperature. The beads were washed 5x in 1 ml of cold PBS. All fluid was removed, and 50 µl of 2x laemmli sample buffer was added or the samples were exposed to PK digestion. Samples were analyzed for PrP levels by SDS-PAGE and Western blot.

Proteinase K digestion and Western blot
Following peptide treatment, cells were extracted in cold lysis buffer. The lysates were cleared by centrifugation for 2 min at 5000 g, and the pellets were removed. Protein concentration was measured by Bradford assay, and protein levels were normalized to 1 mg/ml of protein in all samples. PK digestion was performed on 500 µg of protein as described previously (28) . For PK digestion of heparin-conjugated agarose precipitates, 10 µg of PK was added in a volume of 5 µl lysis buffer without protease inhibitors. Samples were incubated at 37°C for 45 min, then 20 µl of 4x lamaelli sample buffer was added, and samples were boiled for 5 min. The samples were separated on a 12% SDS-PAGE, then transferred to a nitrocellulose membrane and analyzed by Western blot with an anti-PrP antibody as described previously (28) . For immunodetection, enhanced chemiluminescense (ECL) was used.

Statistical analysis
Optical density (OD) measurement was performed on the ECL films using the software Image Gauge V.3.46 (Fuji Photo Film Co. Ltd., Elmsford, NY, USA). OD was measured from Western blots of whole cell lysate to determine values of PrPC or total PrP, or from PK-digested samples to measure levels of PrPSc. In the indicated cases in Supplemental Data, an alternative way of calculating PrPSc levels was used; the OD value of the 18 kDa PrPSc specific band visible on Western blots of whole cell lysate was measured and put in relation to the OD of the total PrP in the sample. PrPC or PrPSc levels in peptide-treated cell cultures were divided by the levels in untreated controls and expressed as percentages. Statistical analyses of relative protein levels were performed using the software GraphPad Prism V4 (GraphPad Software, Inc., San Diego, CA, USA), and graphs for the respective peptides are shown in Supplemental Data. IC50 values for the reducing effect of mPrP1–28 and bPrP1–30 on PrPSc levels in ScGT1–1b cells were calculated using BioDataFit 1.02 (Chang Bioscience, Inc., San Francisco, CA, USA).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
mPrP1–28reduces the PrPSc level in ScGT1–1 cells
Treatment with mPrP1–28 produced a time- and dose-dependent reduction of PrPSc in both ScGT1–1a and ScGT1–1b cells (Figs. 1 B, D and 2 D). A statistically significant reduction of PrPSc levels in ScGT1–1b cells was detected after 3 days of treatment with 1 µM of mPrP1–28, whereas 0.1 µM of mPrP1–28 produced a significant reduction of PrPSc protein levels in ScGT1–1a/b cells after 8 days of treatment (Fig. 2D ). The PrPSc reducing effect caused by mPrP1–28 was quantitatively as efficient as the effect by PPS (Fig. 1A ), which is a well-established antiprion agent. Corresponding treatments with mPrP1–28 in GT1–1 cells resulted in no significant change in PrPC protein levels (Fig. 1C, E ). A scrambled version of this peptide (mPrP1–28R) produced no significant change in the PrPSc levels of ScGT1–1b cells (Fig. 2C and Supplemental Data). Cultivation of ScGT1–1b cells in the presence of 0.5 µM mPrP1–28 showed that this treatment could retain PrPSc at an average 60% reduced level for at least 30 days and if such treatment was aborted, PrPSc returned to the original level after 15 days postpeptide treatment (data not shown).


Figure 1
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  		Figure 1. Time-dependent effect of mPrP1–28 on PrPSc and PrPC protein levels. Treatment with mPrP1–28 for 3, 5, or 8 days. A) Western blot of whole cell lysates from ScGT1–1a cells after treatment with 2 µM mPrP1–28 vs. PPS. Controls are untreated (UT). Reduced levels of PrPSc are clearly visible as lower intensity of the 18 kDa PrPSc band. B) Western blot of PK-treated cell extracts from ScGT1–1a or ScGT1–1b cells after treatment with 2 µM mPrP1–28 vs. UT controls and PrPC in non-PK treated, untreated GT1–1 cell extract (C). C) Western blot of whole cell lysates from GT1–1 cells after treatment with 2 µM of mPrP1–28 vs. UT control. D) Graph showing the relative ratios as percentage of PK-resistant PrPSc in ScGT1–1a or ScGT1–1b cells after treatment with 2 µM mPrP1–28vs. UT control. All treatments show significant reduction of PrPSc levels. *P < 0.05; **P < 0.01; n = 4. E) Graph showing the relative ratios as percentage of PrPC in GT1–1 cells after treatment with 2 µM mPrP1–28 vs. UT control. No significant differences in PrPC levels were found. n = 4


Figure 2
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  		Figure 2. Dose-dependent and PrP-CPP-specific effect of mPrP1–28 and bPrP1–30 on PrPSc levels. Western blots of whole cell extracts (–PK) and PK-treated samples (+PK) from ScGT1–1b cells after 8 days of treatment with peptides mPrP1–28, mPrP23–50, bPrP1–30, mPrP23–38, and penetratin vs. UT ScGT1–1b cells. A) Treatment with 2 µM PrP1–28 results in significantly reduced levels of PrPSc (also seen in Fig. 1 D). However, PrPSc levels are statistically not changed by 2 µM treatment with any of the other peptides. *Final mPrP23–28 concentration 10 µM. B) Treatment of ScGT1–1b cells for 8 days with 10 µM PrP1–28 or bPrP1–30 results in significantly reduced levels of PrPSc. The PrPSc level is not changed by 10 µM treatment with any of the other peptides. *Final mPrP23–28 concentration 50 µM. C) Graph showing the relative levels of PrPSc in ScGT1–1b cells after treatment for 8 days with 5 µM mPrP1–28, mPrP1–28R, mPrP19–30, mPrP23–50, bPrP1–30, or penetratin, or with 25 µM mPrP23–28. PrPSc levels were significantly reduced after treatment with mPrP1–28 or bPrP1–30 but not after treatment with the other peptides. *Final mPrP23–28 concentration 25 µM; **P < 0.01; n = 3. D) Dose-response curves showing that treatment with the PrP-CPPs mPrP1–28 and bPrP1–30 significantly reduced PrPSc levels in ScGT1–1b cells in a dose-dependent manner. PrPSc levels are percentages relative to untreated controls. Peptide concentrations (µM) were tested as presented in Table 1 and are expressed as log10. Calculated IC50 for PrPSc reduction after 8 days of treatment: mPrP1–28, 0.3 ± 0.07 µM; bPrP1–30, 3.3 ± 0.68 µM. n = 3.

bPrP1–30reduces PrPSc levels in ScGT1–1 cells
mPrP1–28 (2 µM) significantly reduced levels of PrPSc in ScGT1–1a/b cells after 3 days of treatment (Fig. 1D ). However, treatment with bPrP1–30 at 2 µM, or lower concentrations, did not produce significant effects even after 8 days (Fig. 2A ). Therefore, 8-day treatments of ScGT1–1b cells were performed with the peptides mPrP1–28, mPrP1–28R, mPrP19–30, mPrP23–50, bPrP1–30, penetratin, and TP-10 at final concentrations of 5 or 10 µM, or with mPrP23–28 at 25 or 50 µM. Presumably, such concentrations could be toxic, since 5 µM of mPrP1–28 results in 50% cellular loss after 20 h treatment of N2a cells (16) . However, any toxicity was efficiently reduced by changing the culture medium on a daily basis, removing any peptide in excess. Treatments of GT1–1 cells with peptides in these higher concentrations showed neither significant cellular loss nor any effect on PrPC protein levels (data not shown). In the concentrations of 5 and 10 µM, bPrP1–30 significantly reduced the levels of PrPSc (Fig. 2B, C ). None of the other tested peptides, except mPrP1–28, had any effect on PrPSc levels, even at these higher concentrations (Fig. 2B, C and Supplemental Data). Comparing the efficiency of the PrP-CPPs in reducing the PrPSc levels in ScGT1–1b cells after 8 days of treatment showed that mPrP1–28 has a IC50 value of 0.3 ± 0.07 µM, whereas bPrP1–30 has a relatively higher IC50 value of 3.3 ± 0.68 µM (Fig. 2D ).

mPrP1–28 and bPrP1–30slow the rate of PrPSc accumulation during early stages of RML infection
GT1–1 cells were infected with scrapie by incubation with RML mouse brain homogenate. During the 3 days of RML treatment, cells were treated with 10 µM of either mPrP1–28, bPrP1–30, mPrP23–50, penetratin, or transportan or with 50 µM of mPrP23–28. After 20 days of cultivation following the 3 days of RML/peptide treatment, the cultures that had been simultaneously incubated with either mPrP1–28 or bPrP1–30 showed significantly lower levels of PrPSc (Fig. 3 A). The course of the RML infection was not affected by any of the other peptides (Fig. 3A, B and data not shown). After the infected cell cultures had been passaged for an additional 15 days (35 days postinfection/peptide treatment), all cultures were propagating PrPSc at similar levels, showing no effect from any previous treatment (Fig. 3B ).


Figure 3
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  		Figure 3. The rate of PrPSc accumulation during early stages of RML infection is slowed by treatment with mPrP1–28 or bPrP1–30. Western blot of whole cell lysates (–) or PK-digested lysates (+) from GT1–1 cells incubated with 0.05% of RML brain homogenate. Cells were extracted 20 or 35 days postinfection. During the 3 days of RML infection, cells were simultaneously treated with 10 µM mPrP1–28, bPrP1–30, penetratin, or transportan-10 (TP-10). As control (C), PBS was added to cells during RML infection. Noninfected GT1–1 cells served as infection control. A) After 20 days of cultivation postinfection, levels of PrPSc were significantly lower in cultures treated with mPrP1–28 or bPrP1–30 during the RML infection period vs. the control, penetratin, and TP-10 treatments. B) When the cultures tested in A were passaged for an additional 3 passages (35 days postinfection), no differences in PrPSc levels were detected.

Binding of PrPC and PrPSc to heparin is not blocked by mPrP1–28
Both PrPC and PrPSc were found to efficiently bind to heparin-conjugated agarose (HA) (Fig. 4 B). PK treatment of such HA precipitates from ScGT1–1b lysates showed a proportion of the precipitated PrP to be PK resistant PrPSc (Fig. 4B , lane +PK). However, preincubation of HA with excess mPrP1–28 (200 µM) prior to the addition of lysate did not affect the levels of precipitated PrPC or PrPSc, compared with the controls (Fig. 4A , HA preinc. mPrP1–28). Likewise, when the lysate was preincubated with excess mPrP1–28 prior to the addition of HA, no change in the levels of PrPC or PrPSc bound to the HA was detected, compared to the controls (Fig. 4A , lysate preinc, mPrP1–28). These results suggest that the antiprion effects of the prion protein-derived peptides do not involve direct interference with the interaction between PrP and HSPGs at the cell surface.


Figure 4
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  		Figure 4. Neither PrPC nor PrPSc binding to heparin is affected by the presence of mPrP1–28. A) Graph displaying levels of PrP in HA precipitates made on GT1–1 or ScGT1–1b cell lysates. Precipitation of PrPC was detected from GT1–1 cells. Precipitates from ScGT1–1b cells were exposed to PK degradation before SDS-PAGE, showing PrPSc binding to HA. Final concentration of mPrP1–28 during incubations, in samples where peptide was added, was 67 µM. For control 1, HA beads were preincubated with lysis buffer before addition of lysate and further incubation. For control 2, lysates were preincubated with lysis buffer before addition of HA beads and further incubation. Preincubation of HA with 200 µM mPrP1–28 had no effect on levels of precipitated PrPC from GT1 or PrPSc (+PK) from ScGT1–1b (lanes HA preinc. mPrP1–28) vs. controls (control 1). Preincubation of lysates with 100 µM of mPrP1–28 prior to HA precipitation had no effect on levels of precipitated PrPC from GT1 or PrPSc (+PK) from ScGT1–1b (lanes lysate preinc. mPrP1–28) vs. controls (control 2). n = 3. B) Western blot detection of PrPSc in HA precipitates after (+) PK digestion vs. PrP in nondigested HA precipitates from ScGT1–1b or GT1–1 lysates.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we present the new finding that peptides derived from the N terminus of the unprocessed prion protein can antagonize prion infection. The PrP peptides that reduce the PrPSc levels in ScGT1–1 cells include the N-terminal signal sequence as well as a basic segment and are known to function as CPPs. mPrP1–28 strongly reduces the PrPSc protein levels in ScGT1–1a/b cells without affecting the PrPC protein levels in GT1–1 cells (Fig. 1) . bPrP1–30has a similar anti-PrPSc effect as mPrP1–28, although somewhat higher concentrations of the peptide are needed (Fig. 2D ). Also, a brief treatment with PrP-CPP significantly slowed the rate of prion accumulation and/or prion formation during the initial phase of RML scrapie infection (Fig. 3A ).

Recently, two peptides derived from the human prion protein, hPrP19–30 and hPrP100–111, were found to interact specifically with PrPSc without any significant binding to PrPC (30) . The interaction was found to be affected by amino acid composition and foremost by the positive charge of the two peptides. We hypothesize that the charged NLS-like sequence may also be responsible for a specific interaction between the PrP-CPPs and PrPSc and that this interaction is important for the reducing effect these CPPs have on the PrPSc levels in ScGT1–1 cells. That the peptide hPrP19–30 was found not to interact with PrPC to any significant extent (30) is in agreement with our present results, in which none of the PrP-CPPs or PrP peptides tested, including mPrP19–30, had any effect on PrPC protein levels in GT1–1 cells.

The reducing effect on PrPSc levels is specific for the CPPs derived from the N terminus of the prion proteins. Recently, our laboratory investigated the importance of the NLS-like sequence, residues 23–28 in mPrP, for internalization by comparing the two peptides mPrP1–28 and mPrP23–50. This study showed that the KKRPKP sequence is not sufficient to cause membrane perturbation or translocation but that this segment needs a hydrophobic counterpart to achieve CPP properties (31) . Here, the hydrophobic signal peptide part of mPrP1–28 provides the CPP activity, whereas the cationic segment, residues 23–28, may be responsible for the actual interaction with PrPSc. The signal sequence may even function as a label for cellular localization of the NLS-like sequence, after promoting cell membrane translocation of the peptide.

We conclude that the antiprion properties of mPrP1–28 and bPrP1–30 are not due strictly to their cationic properties. Other positively charged CPPs tested, such as R16, showed no significant effects on the PrPSc levels; neither did the PrP-derived peptides mPrP23–28, mPrP19–30, and mPrP23–50, which include the same basic residues as the PrP-CPPs (Fig. 2 and Supplemental Data). In the previously published report on hPrP19–30, studies of successively shortened versions of the peptide made it possible to identify residues 23–30 as the peptide core important for binding to PrPSc (30) . Here we show that even if a peptide contains this sequence and the feature of potential PrPSc interaction, this feature alone is still not enough to promote successful antagonization of a prion infection. Even if the cationic domains of these peptides promote association with PrP, if the peptide is not a PrP-CPP the interaction does not take place in a subcellular context, which can affect the PrPSc levels. Hypothetically, the signal sequence segment could guide the peptide into the cell and to a specific cellular compartment, where the basic segment interacts with PrPSc and interferes with its accumulation.

Interestingly, in another recent study, recombinant peptide aptamers, selected from a combinatorial library to have affinity for PrP, were shown to abolish PrPSc conversion when added to prion-infected neuroblastoma cells (ScN2a). Two of these peptide aptamers were also found to interfere with PrPSc formation when expressed in the secretory pathway of ScN2a cells (32) . Although these aptamer peptide sequences have little similarity with the sequences described in the present study, the results suggest that if PrPSc binding motifs can be targeted to the ER secretory pathway, they may interfere with PrPSc conversion in the cell. Other reports on PrP trafficking in the cell suggest that the endocytotic pathway has an important role in the formation of PrPSc (10) . We hypothesize that the PrP-CPPs inhibit conversion along this pathway, which may in part involve the same cellular entities as secretion.

In their study of human PrP sequences, Lau et al. (30) found no capability of the hPrP1–12 peptide to bind to PrPSc from plasma. The strong hydrophobicity of the 1–22 signal peptide by itself renders it unfit for cellular treatment in aqueous solution. Nevertheless, the signal peptide is constantly present in the neuronal cell, through endogenous PrPC expression and processing in the ER, where the signal peptide normally is cleaved off. The possibility that the 1–22 signal sequence alone could reduce PrPSc levels is unlikely. Quite to the contrary, several arguments have been made, that an unprocessed signal peptide in PrP may actually promote PrPSc formation and prion infection (17 , 24 , 31) .

Finally, we would like to consider possible mechanisms to explain the antiprion effect of mPrP1–28 and bPrP1–30 (Fig. 5 ). The PrP-CPPs enter the cell through macropinocytosis (17) . Following macropinocytosis, the PrP-CPPs may become shuttled to the Golgi apparatus and through vesicle transport circle in the secretory pathway. The straightforward interpretation of our results is that the PrP-CPPs bind to PrPSc in a cellular compartment where PrPC to PrPSc conversion takes place. The association of the peptide to PrPSc hinders or prevents further recruitment or conversion of PrPC to the scrapie form (Fig. 5A ). Alternatively, the PrP-CPPs may associate to PrPC, promoting similar effects as described for the peptide aptamers endogenously expressed in the secretory pathway (Fig. 5B ) (32) . However, our present data do not indicate any interactions between the PrP-CPPs and PrPC, which makes this mode of action unlikely.


Figure 5
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  		Figure 5. Potential interfaces for the antiprion action of prion protein-derived peptides. PrP-CPPs mPrP1–28 and bPrP1–30 and their possible routes to conversion interference. A) Binding of the PrP-CPP blocks the interaction site on PrPSc necessary for interaction with PrPC and inhibits conversion, possibly in an endosomal compartment or on the cell surface. B) Lipid raft-mediated cell penetration may result in shuttle of the PrP-CPP to the secretory pathway, where the peptide may interfere with PrPSc formation through overstabilization of PrPC. C) Binding of PrP-CPP to PrPSc may render PrPSc more susceptible to degradation. Also, PrP-CPP induced macropinocytosis may promote an endocytic pathway and/or target a degradation fate enhancing the reduction of PrPSc. D) PrP-CPP interaction with cell surface HSPGs may introduce a steric hindrance to the molecular platform necessary for conversion.

It is possible that introduction of the PrP-CPPs could alter the endocytotic fate of rafts and the resident PrP by initiation of lipid-raft mediated macropinocytosis (17) . This could change the propensity of PrPC to become available for conversion, or it may elevate the degradation of PrPSc. Another possible mechanism underlying their antiprion properties is that the binding of the PrP-CPPs to PrPSc by itself may render PrPSc more susceptible to lysozomal degradation in a yet unknown manner (Fig. 5C ).

HS could be involved in the anti-PrPSc function of PrP-CPPs through several conceivable mechanisms. Since HS chains are shown to facilitate PrPSc conversion in vivo and in vitro (11 , 12) , the PrP-CPPs also interacting with HS might interfere with PrPSc formation by competitive binding to cell surface HSPGs. However, our present data show that mPrP1–28 does not block the binding of heparin-conjugated agarose to PrPC or PrPSc (Fig. 4A , lanes HA preinc. mPrP1–28). Correspondingly, nor does mPrP1–28 block heparin-agarose binding sites on PrPC or PrPSc (Fig. 4A , lanes lysate preinc. mPrP1–28). If indeed interaction between the PrP-CPPs and HS is involved in counteracting PrPSc conversion, mPrP1–28 and bPrP1–30 may introduce steric hindrance into the PrPC-HS-PrPSc complex, disabling the putative function of this as a conversion platform (Fig. 5D ).

In conclusion, the PrP-CPPs composed of the hydrophobic PrP signal peptide and the positively charged NLS-like sequence induce strong antiprion effects in infected cells without disturbing the levels of the normal PrPC. This antiprion activity is not mimicked by other CPPs or by PrP peptides lacking CPP function. The PrP-CPPs are not found to antagonize prion infection by blocking interaction between PrP and HS chains. We conclude that the two segments of the PrP-CPPs (signal sequence and NLS-like segment KKRPKP) provide two functionalities, i.e., cellular entry and localization, as well as specific PrPSc interaction. Both functionalities are necessary for the antiprion effect of these compounds. Our results shed light on the process of prion protein conversion to scrapie form and suggest principles for developing drugs against prion diseases.


   ACKNOWLEDGMENTS
 
We gratefully thank Prof. Stanley B. Prusiner (University of California, San Francisco, CA, USA) for the generous gifts of RML-infected brains and GT1–1 cells. This work was supported by the Swedish Medical Research Council, The Swedish National Board for Laboratory Animals (CFN), The Swedish Research Council for Environment, Agricultural Science and Spatial Planning (FORMAS), and Magnus Bergvalls stiftelse.

Received for publication December 2, 2007. Accepted for publication January 24, 2008.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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