Archives of Biochemistry and Biophysics 564 (2014) 254–261

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Targeting prion propagation using peptide constructs with signal sequence motifs Kajsa Löfgren Söderberg a,⇑, Peter Guterstam b, Ülo Langel b, Astrid Gräslund a,⇑ a b

The Department of Biochemistry and Biophysics, Stockholm University, SE-106 91 Stockholm, Sweden The Department of Neurochemistry, Stockholm University, SE-106 91 Stockholm, Sweden

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Article history: Received 11 September 2014 and in revised form 15 October 2014 Available online 25 October 2014 Keywords: Prion Signal peptide Polycationic motif Cell penetrating peptide

a b s t r a c t Synthetic peptides with sequences derived from the cellular prion protein (PrPC) unprocessed N-terminus are able to counteract the propagation of proteinase K resistant prions (PrPRes, indicating the presence of the prion isoform of the prion protein) in cell cultures (Löfgren et al., 2008). The anti-prion peptides have characteristics like cell penetrating peptides (CPPs) and consist of the prion protein hydrophobic signal sequence followed by a polycationic motif (residues KKRPKP), in mouse PrPC corresponding to residues 1–28. Here we analyze the sequence elements required for the anti-prion effect of KKRPKP-conjugates. Neuronal GT1-1 cells were infected with either prion strain RML or 22L. Variable peptide constructs originating from the mPrP1–28 sequence were analyzed for anti-prion effects, measured as disappearance of proteinase K resistant prions (PrPRes) in the infected cell cultures. We find that even a 5 amino acid N-terminal shortening of the signal peptide abolishes the anti-prion effect. We show that the signal peptide from PrPC can be replaced with the signal peptide from the Neural cell adhesion molecule-1; NCAM11–19, with a retained capacity to reduce PrPRes levels. The anti-prion effect is lost if the polycationic N-terminal PrPC-motif is conjugated to any conventional CPP, such as TAT48–60, transportan-10 or penetratin. We propose a mechanism by which a signal peptide from a secretory or cell surface protein acts to promote the transport of a prion-binding polycationic PrPC-motif to a subcellular location where prion conversion occurs (most likely the Endosome Recycling Compartment), thereby targeting prion propagation. Ó 2014 Elsevier Inc. All rights reserved.

Introduction The physiological role(s) of the cellular prion protein (PrPC) appear to be many and varied [1]. The prion protein is also involved in protein misfolding disorders such as CreutzfeldtJacob’s disease in humans, Bovine Spongiform Encephalitis (BSE)1 in cattle, or Scrapie in sheep, which belong to a family of important neurodegenerative amyloid diseases [2]. The so called Scrapie isoform of the prion protein (PrPSc, in infectious prions), probably in an oligomeric form [3], is a biological and medical concept associated with infection and disease. Here we will generally use the term ⇑ Corresponding authors at: Department of Biochemistry and Biophysics, The Arrhenius Laboratories, Svante Arrhenius Väg 16 C, Stockholm University, SE-106 91 Stockholm, Sweden. Fax: +46 8 155597. E-mail addresses: [email protected] (K.L. Söderberg), [email protected] (A. Gräslund). 1 Abbreviations used: BSE, Bovine Spongiform Encephalitis; ERC, Endosome Recycling Compartment; HSPGs, heparan sulfate proteoglycans; CPPs, cell penetrating peptides; FBS, fetal bovine serum; PBS, phosphate buffered saline; OD, optical density; NCAM1, Neural cell adhesion molecule-1. http://dx.doi.org/10.1016/j.abb.2014.10.009 0003-9861/Ó 2014 Elsevier Inc. All rights reserved.

PrPRes, chemically defined as the isoform of PrP which is resistant to proteinase K, a characteristic closely but not entirely linked to prion infectivity [4,5]. It has recently become clear that other amyloid diseases share important traits with the prion diseases, such as transcellular spread of amyloid material. This makes prion studies relevant also for a wider range of amyloid diseases [6]. While the cellular localization of PrPC varies between cell types, the protein generally follows the secretory pathway (as reviewed in [7]). Most PrPC is found on the plasma membrane, from where it is internalized and travels via early endosomes and recycling endosomes back onto the cell surface [8]. A small fraction of PrPC is internalized for lysosomal degradation [9]. PrPC traffics different uptake pathways depending on membrane subdomain dynamics [10]. Endocytosis is an important key event for prion propagation [11] and the Endosome Recycling Compartment (ERC) has been proposed as a major site of the PrPSc-induced misfolding of PrPC (prion conversion) [8]. Like amyloid conversion in general, the conversion process of PrPC into PrPSc is not well defined chemically.

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It appears to depend on PrPC and/or PrPSc membrane insertion [12], and oligomeric PrPSc nucleating misfolding of PrPC [13–15]. The efficiency of prion replication is affected by several proposed environmental cofactors [16] such as pH and presence of ions, as well as the presence of cell surface heparan sulfate proteoglycans (HSPGs) [17,18]. The gradually lowered pH (pH in the range 4.7–5.8) of maturing endosomes is a potential driving force for prion conversion [11] [19,20]. Although the ERC has been proposed as a major site for prion replication [8,21,22], other conversion sites also may exist, such as the plasma membrane [23]. While membrane rafts control prion uptake [10], neuronal cell surfaces in BSE infected cattle exhibit abnormal coated pit formations associated with presence of misfolded PrP [24]. Membrane proteins interacting with PrPC/ PrPSc in both raft and non-raft membrane domains may promote their endocytosis [10,25] and possibly also their toxicity [26]. Pinocytosis of prions has also been reported [27,28]. The prion conversion site(s) may vary between different cells [22,29–33], and most likely depends on factors like prion strain, PrPSc-subpopulations [34–36] as well as uptake routes. The cell penetrating peptides (CPPs) are a heterogeneous group of peptide constructs, which are related to but generally less cytotoxic than antimicrobial peptides. CPPs have the capacity to internalize into cells and also bring with them various attached cargoes. CPPs have two proposed mechanisms for cellular uptake: by triggering an active uptake and possibly by passive transduction directly across the cell membrane [37]. The active endocytotic uptake may follow different pathways, leading to endosomal escape into the cytoplasm [38]. Peptides derived from the unprocessed prion protein N-terminus can cross biological membranes also when complexed with cargoes [39–41]. These CPP-like PrP-peptides contain the prion protein hydrophobic signal peptide (mouse PrP residues 1–22), normally removed from PrPC during biosynthesis, followed by a polycationic nuclear localization-like sequence, the NLS-like sequence (mouse PrP residues 23–28; KKRPKP). The membrane translocation of such a peptide with a sequence derived from the mouse or bovine PrP (mPrP1–28 or bPrP1–30, see Table 1) was suggested to occur through raft-dependent macropinocytosis, initiated/mediated by cell surface HSPGs and/or negatively charged phospholipids, followed by endosomal escape [41]. We have previously reported that externally added mPrP1–28 and bPrP1–30 significantly down-regulate PrPRes (proteinase K resistant PrP) levels in prion-infected cells [42]. To further analyze the biochemical background for this anti-prion effect, our present work explores the effects of varying peptide constructs added to prion infected cell cultures. The peptide constructs contain the polycationic segment KKRPKP, expected to bind to PrPRes [43,44]. The polycationic sequence is coupled to different CPPs, or to an alternative signal peptide targeting the ER for secretary proteins (from the Neural cell adhesion molecule-1; NCAM1-19) (Table 1). While the constructs based on CPP sequences had no effect on PrPRes, the construct based on the NCAM-1 was even more efficient than the original mPrP1–28 to reduce the PrPRes levels.

Materials and methods Cell cultures and RML infection The GT1-1 cell line [45] is derived from immortalized murine gonadotropin-releasing hypothalamic neuronal cells. Cells were cultivated in Dulbecco’s modified Eagle’s medium 4.5 g/L glucose with Glutamax I (DMEM) supplemented with 5% heat-inactivated fetal bovine serum (FBS), 5% heat-inactivated horse serum (HS), and 50 U/ml penicillin–streptomycin (PEST). Confluent cell

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cultures were split at a ratio of 1:5 once a week using 1X-trypsin–EDTA (Gibco BRL). Infection of GT1-1 cells with RML/Chandler prion isolate to generate ScGT1-RML cells was performed as previously [46], and of of GT1-1 cells with 22L prion isolate to generate ScGT1-22L cells [46], by using 0.1% homogenate of mouse brains infected with the prion strain. The RML/Chandler isolate homogenate was a generous gift from Prof. Stanley B. Prusiner (Institute for Neurodegenerative Diseases and Department of Neurology, University of California, San Francisco, CA, USA). The 22L isolate homogenate was obtained from The Roslin Institute, University of Edinburgh, Scotland. Both ScGT1cell lines were regularly tested for presence of PrPRes.

Peptide synthesis Peptide synthesis was performed on a SYRO multiple peptide synthesizer (MultiSyn Tech Gmbl) using a polystyrene-based Rink amide resin (0.4–0.6 mmol/g). Standars Fmoc (9 H-fluoren-9ylmethoxycarbonyl)-AA-OH were coupled using HBTU (O-benzotriazole-N, N, N0 , N0 -tetramethyl-uroniumhexafluorophosphate) as activating reagent and DIEA (diisopropylethyl amine) as base. Peptides were purified by RP-HPLC, C18 preparative column (5 lm), acetonitrile–water [0.1% TFA] and analyzed by MALDI MS (Perkin-Elmer prOTOF™ 2000 O-TOF, positive mode).

Peptide treatments of cell cultures The peptide constructs, as listed in Table 1, were tested for effects on PrPC levels in GT1 cells and effects on PrPRes in cells infected with either of prion strains RML/Chandler or 22L. Before treatment of cells with the respective peptide, the GT1, ScGT1RML, or ScGT1-22L cells were seeded on 12-well Petri cell culture plates in HS/FBS/PEST-supplemented DMEM. 0.5 ⁄ 106 cells /well were seeded out 7 days before harvest and analysis of prion protein levels. Peptides were added at a final concentration of 5 lM if not stated otherwise, in HS/FBS/PEST-supplemented DMEM. Equivalent addition of phosphate buffered saline (PBS) was used for negative controls (UT C). All treatments were conducted over 5 days. Cells were subjected to two additions of indicated peptide, at 5 and 3 days prior to cell harvest, and medium was changed prior to each peptide addition.

Immunodetection of prion protein Following peptide treatment, cells were washed thrice in ice cold PBS, trypsinated and washed in PBS by centrifugation at 200g for 5 min. Cell pellets were dissolved in ice cold extraction buffer (0.5% Triton X-100, 0.5% NaDoc, 150 mM NaCl, 10 mM EDTA, 50 mM Tris pH 7.5 at 0 °C). Debris was removed after centrifugation for 2 min at 5000g at 4 °C. Protein content was measured by Bradford assay and samples diluted to achieve equal protein concentration in samples of a given experiment. Each sample was split into two parts; one part cell extract and one part to be subjected to proteinase K (PK)-digestion. For cell extracts, samples were boiled for 5 min in 1/4 final volume of 4xLaemmli sample buffer. For PK-digestion, samples were prepared as previously described [46]. Samples were applied on 12% SDS–PAGE or 4–12% bis-MES NuPAGEÒ gels followed by transfer onto PVDF membrane. Following transfer, each membrane was stained with ponceau-S solution. Analysis by Western blot was performed as described previously [47]. For immunodetection, enhanced chemiluminescense (ECL) was used and images recovered in a LAS-1000 Luminescent Image Analyzer V2.6 (Fuji Photo Film Co., Ltd) using the Fujifilm software MultiGauge V3.2.

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Statistical analysis Densitometry quantifications of PrPC, PrPRes or total prion protein (PrPTot) as presented in staple diagrams were performed essentially as described earlier [18,42], and involve the following steps (exemplified in Fig. S1): optical density (OD) measurement was performed using the software Image Gauge V.3.46 (Fuji Photo Film Co. Ltd., Elmsford, NY, USA). The OD-signal, as measured in arbitrary units (AU) was measured from Western blot ECL-immunodetections for each sample, in a square area between 0 and 50 kDa to enclose all PrP glycosylation variants and PrPRes specific bands. Background signals were measured to give contributions in the range of 1:1000–10,000 compared to PrP-signals. Levels of PrPC (from GT1 cells) or PrPTot (from ScGT1 cells) were measured from cell extract samples. From these blots, OD was also measured from a ponceau stain in a square area between 30 and 50 kDa, to determine a relative ratio of protein concentration between samples. Quantification of PrPRes was measured from PK-digested samples. For cell extract samples, PrP-signals were adjusted to account for the ponceau ratio and for small background signals. For PKdigested samples, the PrPRes signal AU values were also adjusted according to the ponceau of corresponding cell extracts. To enable comparison of PrPRes levels between gels and experiments, a PK-digested GT1 sample loaded on each gel was used. Levels PrPC, PrPRes or total PrP in samples from peptide-treated cells were set in percentage of untreated controls. All peptide treatments were repeated at least 3, or up to 7 times. Statistical analysis was done in GraphPad Prism V5 (GraphPad Software, Inc., San Diego, CA, USA). Materials Cell culture plates were purchased from Invitrogen AB (Lidingö, Sweden) and all cell culture reagents were obtained from Gibco BRL (Paisley, UK). Peptide sequences including terminal modifications are listed in Table 1. Penetratin, transportan-10 (TP10), TP10-mPrP23–28, mPrP6–28 and mPrP12–28 were synthesized as described here. Peptides mPrP1–28 in D-configuration and peptide mPrP1–22L23–28D were obtained from CPPEP Eesti (Tallin, Estonia). All other peptides were purchased from Neosystem Laboratoire (Strasbourg, France). Scrambled mPrP1–28R (randomized) peptide was designed as previously described [48]. Bradford reagens was from Bio-Rad (Hercules, CA, USA) Polyclonal PrP (M-20) antibody and monoclonal S20a1 antibody and secondary donkey anti-goat peroxidase-conjugated antibody were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Alexa fluor 594, Orange green 488, FITC and Texas Red conjugated secondary antibodies together with NuPAGE precast SDS–PAGE gels and Western blotting buffers were from Invitrogen AB. ECL Advance Western Blotting Detection Kit was from GE Healthcare Life Sciences (Pittsburgh, PA, USA). Monoclonal PrP antibody IPC1 and all other reagents were from Sigma–Aldrich Sweden AB (Stockholm, Sweden). Results mPrP1–28 counteracts both RML and 22L prion infection The mPrP1–28 effects were compared for two different prion strains. Prion strains RML and 22L differ in infectivity in cell lines and trigger various changes in gene expression and pathological hallmarks in infected mice [46]. Here, proteinase K (PK)-resistance was used as a diagnostic of formation of the Scrapie form of PrP [4,5]. Western blots were used to detect total PrP-levels (PrPTot) in cell extracts (Fig. 1A, B and D; left panels) and PrPRes in PK-digested samples for the two prion-infected GT1 cell lines

ScGT1-RML and ScGT1-22L (Fig. 1A and B; right panels). The optical density (OD) of each sample signal was measured and correlated to a ponceau stain of the blot with cell extract samples (see Fig. S1 for the data retrieval procedure). Treatment with mPrP1–28 produced a reduction of PrPRes in both ScGT1-RML (Fig. 1A) and ScGT1-22L cell lines (Fig. 1B), also shown in Fig. 1C. The effect was dose dependent and present after treatment for 5 days in 1 lM of mPrP1–28 for both ScGT1 cell lines (data not shown). mPrP1–28 treatment did not affect PrPC levels in uninfected GT1 cells (Fig. 1D). The PrPC-signal remained unchanged also in the ScGT1 cells (Fig. 1A and B, left panels, c.f. C left panel). mPrP1-28 anti-prion effect is not dependent of peptide configuration mPrP1-28 was constructed in three different chiral configurations (Table 1). All three cell lines were treated with mPrP1-28 in L- or D-configurations, denoted mL and mD, respectively, or with a mPrP1-28 peptide where residues 1–22 were in D- and residues 23–38 (i.e., KKRPKP) were in L-form (mPrP1–22D23–28L), denoted mDL. 5-day treatments with either of these constructs resulted in significant reductions of PrPRes in both RML- and 22L-infected GT1 cells (Fig. 1A, C and D). Both the D-form of mPrP1–28 and mPrP1–22D23–28L were quantitatively at least as efficient as the L-form in lowering PrPRes levels in both infected cell lines, and observed already after 5-day treatments with 1 lM of peptide for both prion strains (data not shown). Corresponding treatments of GT1 cells with any mPrP1–28 construct showed no effect on the PrPC protein levels (Fig. 1B and E). The efficient anti-prion effects of the peptides containing Damino acids strongly suggest that no chiral PrP-CPP receptor is involved in the effects of the peptides on the PrPRes levels. Signal peptides direct the polycationic PrP-motif to a cellular site where prions are targeted Translation of PrP into the secretory pathway is directed through its N-terminal signal sequence. Despite a significant sequence difference to mPrP1-28 in the signal peptide motif, the corresponding peptide constructed from the bovine prion protein, bPrP1–30 (Table 1), has similar anti-prion effects on the PrPRes levels in 22L-infected cells (Fig. 2B and D). As most GPI-anchored proteins follow the same cellular processing [49], we investigated if other signal peptide motifs of similar proteins may have functions similar to mPrP1–22. One such signal sequence is present in the Neural cell adhesion molecule-1 (NCAM1), a PrPC-interacting protein [50]. We constructed a chimeric peptide where the signal peptide NCAM11–19 was conjugated to mPrP23–28. The anti-prion effect from this construct was very potent in both RML- and 22L-infected cultures (Fig. 2). We therefore concluded that the polycationic N-terminal PrPmotif KKRPKP should be responsible for the anti-prion effect of mPrP1–28, as residues 1–22 can replaced by another signal peptide motif. The observation also indicates a novel function of a signal peptide when attached to hydrophilic moiety (to increase solubility) from outside a cell: the whole construct is directed to the cellular secretory system. To further investigate if the signal peptide segment is necessary for the function of mPrP1–28 to decrease PrPRes levels in prion infected cells, a peptide was constructed lacking the N-terminal residues 1–11 (Table 1). Treatments with the shortened peptide, mPrP12–28, failed to produce any significant effect on PrPRes levels in either infected cell line (Fig. 2). Even a 5 amino acid shortening of the signal peptide into the construct mPrP6–28 gave a peptide without the anti-prion effects observed for the complete constructs in both infected cell cultures (Fig. 2). Similarly, treatments with a peptide construct solely encompassing the PrP motif, residues

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Fig. 1. Effects of mPrP1–28 on levels of RML and 22L-prions. Prion protein levels in scrapie-infected and uninfected cells after treatment with 5 lM of indicated configuration of mPrP1–28 peptide for 5 days, as compared to untreated controls (UT). Western blots showing PrP-levels in samples from (A) ScGT1-RML cells and (B) ScGT1-22L cells. Left upper panels; PrPTot levels in whole cell extracts applied on 12% SDS–PAGE gels. In these non-PK-digested (-PK) samples, both PrPC and PrPRes is present. Left lower panels; ponceau staining showing the overall protein levels in the whole cell extracts. Right panels; Levels of PrPRes in proteinase K (PK) treated samples, which were run on 4–12% NuPAGE gels. All PrPC has been digested and is no longer present in the samples. (C) Graph showing the relative levels of PrPRes in ScGT1-RML and ScGT1-22L cells respectively, after treatment with 5 lM of indicated configuration of mPrP1–28 for 5 days. The staples show the mean level of PrPRes with bars representing the range of measured values. All treatments show significant reduction of PrPRes levels in both cell lines as compared to the untreated control (UT). Number of experiment replicates (n) = 4. Measurements and calculation procedures are described in Fig. S1 and in the methodological section. (D) Left panel; Western blot of PrPC levels in non-PK-digested (PK) whole cell extracts from uninfected GT1-1 cells after treatment with 5 lM of indicated peptide configuration for 5 days and Right panel; corresponding graph. No differences in PrPC levels were detected n = 6. PrP M20 (Santa Cruz) antibody was used for blots of whole cell extracts, while monoclonal IPC1 antibody (Sigma Aldrich) was used for blots with PK-digested samples. UT; untreated control: mL; mouse (m) PrP1–28 L-form: mD; mPrP1–28 D-form: mDL; peptide mPrP1–22L23–28D. Statistical significance ⁄⁄ P 0.01; ⁄⁄⁄P 0.001.

23–28 (KKRPKP), did not affect PrPRes levels in in either infected cell culture (data not shown), confirming our previous results [42]. Corresponding treatments of GT1-1 cells with the shortened constructs did not result in any significant change in PrPC protein levels (Fig. S2). The lack of anti-prion effects by mPrP6–28 (and mPrP12–28 as well as mPrP23–28) suggests that mPrP1–28 must translocate into the cell to affect prion-infection, and that this requires an intact signal peptide motif. The CPPs Penetratin, Transportan-10 (TP10) and Tat48–60 are able to carry conjugated cargoes across cellular membranes. The Tat48–60 sequence originates from the transactivator of transcription (TAT) protein of the human immunodeficiency virus (see Table 1) [51–53]. Penetratin is an amphiphatic CPP derived from the Drosophila Antennapedia homeodomain protein [54]. TP10 is a chimeric peptide derived from an antimicrobial peptide linked to the N-terminus of the neuropeptide galanin. The translocation mechanisms vary depending on conditions for all CPPs [55–58], and include receptor-dependent endocytotic uptake mechanisms as well as macropinocytosis and possibly direct transduction across cellular membranes. In our further experiments, peptide constructs were made to include the KKRPKP motif conjugated to either Penetratin, Tat48–60 or TP10 (Table 1). We found that none of these constructs gave any significant changes in PrPRes levels of the infected cells (Fig. 2). We also confirmed that the infected cells did not show any reduction of PrPRes levels in response to either Tat48–60 (Fig. 2A, C and D), or to Penetratin or TP10 treatment, confirming previous results [42]. Corresponding treatments of

uninfected GT1-1 cells with any of these constructs did not result in any effect on PrPC levels (Fig. S2 or as previously shown [42]). We concluded that conjugation to a signal peptide of a secreted membrane protein seems to be necessary and sufficient to keep the anti-prion effect of the KKRPKP motif. The anti-prion effect is not due to increased proteasomal activity The ubiquitin–proteasome-system (UPS) is implicated in prion pathology and PrPSc has a direct, possibly inhibitory, interaction with the proteasomal 20S core particle [59]. To evaluate if peptide treatment could affect PrPRes by enhancing the UPS activity, levels of 20Sa1 were analyzed (Fig. 3). Extracts from ScGT1-RML cells treated with mPrP1–28, NCAM11–19-PrP23–28, or other constructs outlined in Table 1, were subjected to Western blotting against the proteasomal S20a1 subunit. All peptide treatments showed small elevations in the levels of S20a1 – but also peptides with no effect on PrPRes levels produced this response. The result indicates that while CPPs in general might trigger a slightly elevated proteasomal activity in a cell, this does not correlate to the anti-prion effect of the signal peptide-KKRPKP-constructs. Discussion The N-terminal polycationic segment PrPC23–27, KKRPK, has been found to be a self-interaction domain of PrPC [60]. The peptide

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Fig. 2. Effects of peptide constructs on levels of RML- and 22L-prions. Levels of PrPRes in scrapie-infected cells after 5 day treatment with 5 lM of indicated peptide construct, as enlisted in Table 1, as compared to untreated controls (UT). Western blots showing PrPRes in samples from (A) ScGT1-RML cells and (B) ScGT1-22L cells. Upper panels; PrPRes levels in proteinase-K (PK)-digested samples applied on 4–12% NuPAGE gels. Mid panels; PrPTot levels in whole cell extracts applied on 12% SDS–PAGE gels. In these non-PKdigested (-PK) samples, both PrPC and PrPRes is present. Lower panels; Ponceau staining showing the overall protein levels in corresponding cell extracts. Monoclonal IPC1 antibody (Sigma Aldrich) was used. (C) Graph showing the relative levels of PrPRes in ScGT1-RML cells and (D) graph showing the relative levels of PrPRes in ScGT1-22L cells respectively, after treatment with 5 lM of indicated peptide construct for 5 days. Besides the documented anti-prion effect of mPrP1–28 and bPrP1–30 [41], only the treatment with NCAM11–19mPrP23–28 produced a significant reduction of PrPRes levels as compared to the untreated control (UT). This effect was significant for both cell lines. n = 3 for all peptides except mPrP12–28 for which n = 2. No construct had effect on PrPC levels in either GT1 cell cultures or in the scrapie infected cell lines (Fig. S2, 4 and 5). Quantification procedures were the same as described for Fig. 1. The staples show the mean level of PrPRes with bars representing the range of measured values. TP10; transportan-10. Statistical significance ⁄⁄P 0.01; ⁄⁄⁄P 0.001.

Fig. 3. Elevation of proteasomal subunits upon peptide treatments. Western blot showing levels of S20a1 proteasomal subunit in ScGT1-RML extracts after treatment with 5 lM of indicated peptide construct, as enlisted in Table 1, for 5 days, as compared to untreated controls (UT). The ponceau staining reflects the overall protein levels in the samples, from which it is visible that the UT and mPrP6– 28 samples both have somewhat higher protein levels than the other loaded onto this gel.

PrP19–30 is reported to bind to PrPSc but not to PrPC [43], and PrPC23–28 binds to various other amyloid-forming proteins [44]. The N-terminal polycationic PrP-motif is important for the toxicity of several

mutant prion protein isoforms [61] and also regulates the uptake of PrPC [8]. The segment PrP23–31 determines the efficiency of prion formation via a direct interaction to PrPSc, and deletion of residues 23–32 in PrPC greatly extends the life span of RML-infected mice [62]. However, deletion of residues 23–30 also reduces the native folding efficiency and a-helical conformational stability of PrPC [63]. Taken together, these observations in the literature suggest a special structural prion-promoting role for the polycationic N-terminal segment of an intact PrPC. In contrast, the present results suggest that the isolated peptide segment mPrP1–28 provides a steric hindrance for prion conversion. Due to the high affinity of the KKRPKP motif for PrPSc [43], mPrP1–28 may become incorporated into PrPRes oligomers, providing a block for further growth of PrPRes. It is likely that the N-terminal polycationic region of the signal peptide-KKRPKP (s.p.-KKRPKP) constructs may bind to oligomeric amyloid aggregates [43,44], which in the case of prions represent the most infectious prion particles [14]. Where can PrPRes be targeted by the s.p.-KKRPKP constructs? A KKRPKP-motif conjugated to CPP-constructs, designed to rapidly escape to the cytosol after endosome uptake [64], does not reduce PrPRes levels. Similarly, mPrP23–28 alone, or the truncated peptides mPrP6–28 or mPrP12–28, does not reduce the PrPRes levels (Fig. 2).

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Table 1 Amino acid sequences of peptide constructs screened for effects on scrapie prion protein levels. Sequences presented include amidation (NH2). mPrP1–28R (randomized) is a scrambled peptide. All peptides were analyzed in the L-configuration if not stated otherwise.

CPP: cell penetrating peptide; NLS: nuclear localization signal.

PrPC as well as PrPSc in the infected cells have been shown to reside both on the plasma membrane, in the ER and ERC to variable but significant degrees [8]. While prion conversion may occur on the plasma membrane [23], our results with externally added peptides without anti-prion effects suggest that the s.p.-KKRPKP peptide does not have its major effect at the membrane surface, but rather in ER or in an endosomal compartment such as ERC. Our previous studies have shown that the s.p.-KKRPKP construct derived from bovine PrP can trigger macropinocytotic uptake into cells, and after internalization resides mainly in endocytotic vesicles [41]. What is the role of the signal peptide segment? The sequences considered here generally target newly synthesized proteins for secretion, via the ER. These signal sequences have a short basic N-terminal flank, a hydrophobic central segment, followed by a polar neutral C-terminus [65]. We observed that removing the first five residues of the signal sequence abolishes the anti-prion effect of mPrP1–28 (Fig. 2), whereas replacing mPrP1–22 with NCAM11–19 retains, and even seems to enhance, the anti-prion effect. This is in contrast to normal CPPs, which do not promote any anti-prion effect when coupled to KKRPKP. Hydrophobicity scale values along the peptide primary structures (Fig. S3) show that the signal peptides consistently display a relatively large hydrophobic core region in the center of the peptide. The CPPs are more variable in this respect. Whereas TAT48–60 is hydrophilic, TP10 is more hydrophobic and reminiscent of the signal peptides regarding the hydrophobicity pattern. However, one difference between the signal peptides and TP10 is that the signal peptides contain a Trp residue flanking the hydrophobic segment and there is no Trp in TP10. Trp is known to be important for peptide-membrane interactions [66]. Hydrophobicity and the presence of aromatic residues, particularly Trp, could affect the membrane interactions and possibly the fate of the construct residing in the maturing endosome. The two peptide categories may also respond differently to the pH-drop in maturing endosomes, or may end up in different types of endosomes. We propose that the signal peptide segment specifically

triggers transport to a particular subcellular location of its attached cargo when added from the outside to a cell, and consider it likely that this location is the ERC. The endosomal recycling from a macropinosome is rapid [67], and infection of neurotoxic prions through induction of macropinocytosis [28] may trigger conversion in the ERC [8]. Since mPrP1–28 retains its anti-prion effect regardless of chiral configuration (Fig. 1A–C) this is consistent with a macropinocytotic uptake also of the peptide [67]. Both PrPSc and the s.p.-KKRKPK peptides may trigger macropinocytosis. Potential HSPG and membrane interactions may promote interactions between the peptide and PrPScoligomers. Parallel internalization of both peptide and PrPSc would then promote steric hindrance of further prion conversion by the peptides, with ERC as a likely major site for the intervention. The construction of signal peptide:NLS-like motifs might also be used as a general guideline to target other amyloid processes. PrPC traps b-amyloid oligomers [68] and acts as a receptor to mediate toxicity through an uptake involving both Low density lipoprotein related protein-1 (LRP-1) and membrane raft dynamics [26] similar to prion infection [25]. The KLVFF-motif of b-amyloid (Ab16–20) can bind b-amyloid fibrils and arrest their aggregation [69]. The efficiency of this sequence, and related b-sheet breaker peptides [70] could possibly be enhanced if the aggregation-inhibiting peptides were conjugated to signal peptide motifs. Our present observations support earlier proposals of the ERC as a cellular place of prion conversion Fig. 4 shows a scheme where the KKRPKP-motif is guided by a signal sequence segment and internalized to reach an intracellular location, the ERC, [8], where the peptide prevents conversion by binding of the NLS-like polycationic motif to PrPSc. Thereby further growth of PrPSc is prevented. Based on the likely localization of the anti-prion effects to the ERC, the model (Fig. 4) could also present a hypothetic idea for how the ERC might be targeted from the outside of a cell, using signal peptides segments carrying different kinds of more hydrophilic (cationic?) cargoes.

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Fig. 4. Model for blocking prion replication by s.p.-KKRPKP peptides. (A) Association of PrPC (green) to membrane rafts can be facilitated by heparan sulfate proteoglycans (HSPG, purple) [71]. Membrane rafts are displayed as a thicker membrane (orange). Association of PrPC to raft formations may trigger caveolar or caveolar-like uptake. In a healthy cell, endosomal vesicles fuse with early endosomes and PrPC is recycled back onto the plasma membrane. In prion disease, PrPSc interaction with HSPGs and/or its complexed proteins associates PrPSc (orange rhombs) with rafts. Following endocytosis, prion conversion may occur in endosomal compartments destined for recycling back to the plasma membrane (ERC). Here, PrPSc may utilize cofactors to facilitate conversion of PrPC (green rhomb), supported by an endosomal drop in pH. Misfolded PrPSc is trafficked to the proteasome or lysosome or extracellular space for plaque inclusion [72]. (B) PrPC also exists in non-raft membrane domains. PrPC interaction with e.g. Low density lipoprotein related protein-1 (LRP-1, blue) and/or other non-raft proteins, could promote a non-raft association of PrPC upon HSPG release of PrPC [25], followed by clathrin-mediated endocytosis. Endocytic uptake of PrPSc could also occur via caveolar/-like routes and via clathrin coated pits, mediated by LRP-1 interaction [25,73,74]. This uptake pathway can also sustain prion replication. (C) PrPSc oligomers may infect via macropinocytotic uptake [28,41]. The resulting macropinosome, containing both raftand non-raft-associated conversion cofactors, or following endosomal vesicles, may sustain prion conversion. (D) Pathogenic conversion of PrPC to PrPSc. (E1) The membrane binding properties of the signal peptide segment of s.p.-KKRPKP peptides (red/green spiral) induces a non-receptor mediated macropinocytotic uptake [28,41]. (E2) The KKRPKP segment of the peptides interacts specifically with PrPSc and the peptides are incorporated onto the PrPSc-oligomer. This inhibits conversion by blocking the interaction between PrPSc and PrPC. Depicted protein and membrane scales are not interrelated.

Acknowledgments We would like to thank Prof. Krister Kristensson, Karolinska Institutet and Assistant Prof. Katrin Mani, Lund University, for the extensive use of laboratory and microscope facilities.

[5] [6] [7] [8] [9] [10]

Appendix A. Supplementary data [11]

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.abb.2014.10.009.

[12] [13]

References [1] R. Linden, Y. Cordeiro, L.M. Lima, Cell. Mol. Life Sci. 69 (2011) 1105–1124. [2] M. Vendruscolo, T.P. Knowles, C.M. Dobson, Cold Spring Harb. Perspect. Biol. 3 (2011). [3] P. Huang, F. Lian, Y. Wen, C. Guo, D. Lin, Acta Biochim. Biophys. Sin. (2013) (Shanghai). [4] R. Race, A. Jenny, D. Sutton, J. Infect. Dis. 178 (1998) 949–953.

[14] [15] [16] [17] [18]

G. Sajnani, J.R. Requena, Prion 6 (2012) 430–432. M. Jucker, L.C. Walker, Nature 501 (2013) 45–51. V. Lewis, N.M. Hooper, Front. Biosci. 16 (2011) 151–168. Z. Marijanovic, A. Caputo, V. Campana, C. Zurzolo, PLoS Pathog. 5 (2009) e1000426. C. Sunyach, A. Jen, J. Deng, K.T. Fitzgerald, Y. Frobert, et al., EMBO J. 22 (2003) 3591–3601. D. Sarnataro, A. Caputo, P. Casanova, C. Puri, S. Paladino, et al., PLoS One 4 (2009) e5829. D.R. Borchelt, A. Taraboulos, S.B. Prusiner, J. Biol. Chem. 267 (1992) 16188– 16199. G.S. Baron, K. Wehrly, D.W. Dorward, B. Chesebro, B. Caughey, EMBO J. 21 (2002) 1031–1040. C. Kim, T. Haldiman, K. Surewicz, Y. Cohen, W. Chen, et al., PLoS Pathog. 8 (2012) e1002835. J.R. Silveira, G.J. Raymond, A.G. Hughson, R.E. Race, V.L. Sim, et al., Nature 437 (2005) 257–261. K.C. Chen, M. Xu, W.J. Wedemeyer, H. Roder, Biophys J 101 (2011) 1221–1230. K. Abid, R. Morales, C. Soto, FEBS Lett. 584 (2010) 2409–2414. L. Horonchik, S. Tzaban, O. Ben-Zaken, Y. Yedidia, A. Rouvinski, et al., J. Biol. Chem. 280 (2005) 17062–17067. K. Löfgren, F. Cheng, L.Å. Fransson, K. Bedecs, K. Mani, Eur. J. Neurosci. 28 (2008) 964–972.

K.L. Söderberg et al. / Archives of Biochemistry and Biophysics 564 (2014) 254–261 [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] [37] [38] [39] [40] [41] [42] [43] [44] [45] [46]

R. Gerber, A. Tahiri-Alaoui, P.J. Hore, W. James, Protein Sci. 17 (2008) 537–544. W.Q. Zou, N.R. Cashman, J. Biol. Chem. 277 (2002) 43942–43947. X. Li, M. DiFiglia, Prog. Neurobiol. 97 (2012) 127–141. S.F. Godsave, H. Wille, P. Kujala, D. Latawiec, S.J. DeArmond, et al., J. Neurosci. 28 (2008) 12489–12499. R. Goold, S. Rabbanian, L. Sutton, R. Andre, P. Arora, et al., Nat. Commun. 2 (2011) 281. C. Ersdal, C.M. Goodsir, M.M. Simmons, G. McGovern, M. Jeffrey, Neuropathol. Appl. Neurobiol. 35 (2009) 259–271. A. Jen, C.J. Parkyn, R.C. Mootoosamy, M.J. Ford, A. Warley, et al., J. Cell Sci. 123 (2010) 246–255. J.V. Rushworth, H.H. Griffiths, N.T. Watt, N.M. Hooper, J. Biol. Chem. 288 (2013) 8935–8951. S. Sabharanjak, P. Sharma, R. Parton, S. Mayor, Dev. Cell 4 (2002) 411–423. J.S. Wadia, M. Schaller, R.A. Williamson, S.F. Dowdy, PLoS One 3 (2008) e3314. J.E. Arnold, C. Tipler, L. Laszlo, J. Hope, M. Landon, et al., J. Pathol. 176 (1995) 403–411. S.J. Barmada, D.A. Harris, J. Neurosci. 25 (2005) 5824–5832. B. Caughey, G.J. Raymond, D. Ernst, R.E. Race, J. Virol. 65 (1991) 6597–6603. M.P. McKinley, A. Taraboulos, L. Kenaga, D. Serban, A. Stieber, et al., Lab. Invest. 65 (1991) 622–630. F. Pimpinelli, S. Lehmann, I. Maridonneau-Parini, Eur. J. Neurosci. 21 (2005) 2063–2072. H. Kretzschmar, J. Tatzelt, Brain Pathol. 23 (2013) 321–332. M. Jeffrey, G. McGovern, C.M. Goodsir, S. Siso, L. Gonzalez, Brain Pathol. 19 (2009) 1–11. C. Weissmann, J. Li, S.P. Mahal, S. Browning, EMBO Rep. 12 (2011) 1109–1117. F. Madani, S. Lindberg, Ü. Langel, S. Futaki, A. Gräslund, J. Biophys. (2011). 414729. E. Koren, V.P. Torchilin, Trends Mol. Med. 18 (2012) 385–393. P. Lundberg, M. Magzoub, M. Lindberg, M. Hällbrink, J. Jarvet, et al., Biochem. Biophys. Res. Commun. 299 (2002) 85–90. H. Biverståhl, A. Andersson, A. Gräslund, L. Mäler, Biochemistry 43 (2004) 14940–14947. M. Magzoub, S. Sandgren, P. Lundberg, K. Oglecka, J. Lilja, et al., Biochem. Biophys. Res. Commun. 348 (2006) 379–385. K. Löfgren, A. Wahlström, P. Lundberg, Ü. Langel, A. Gräslund, et al., FASEB J. 22 (2008) 2177–2184. A.L. Lau, A.Y. Yam, M.M. Michelitsch, X. Wang, C. Gao, et al., Proc. Natl. Acad. Sci. U.S.A. 104 (2007) 11551–11556. A. Yam, X. Wang, C. Gao, M. Connolly, R.N. Zuckermann, et al., Biochemistry 50 (2011) 4322–4329. S.P. Mahal, C.A. Baker, C.A. Demczyk, E.W. Smith, C. Julius, et al., Proc. Natl. Acad. Sci. U.S.A. 104 (2007) 20908–20913. P.J. Skinner, H. Abbassi, B. Chesebro, R.E. Race, C. Reilly, et al., BMC Genomics 7 (2006) 114.

261

[47] H. Gyllberg, K. Löfgren, H. Lindegren, K. Bedecs, FEBS Lett. 580 (2006) 2603– 2608. [48] Ü. Langel, T. Land, T. Bartfai, Int. J. Pept. Protein Res. 39 (1992) 516–522. [49] Y. Maeda, T. Kinoshita, Prog. Lipid Res. 50 (2011) 411–424. [50] G. Schmitt-Ulms, G. Legname, M.A. Baldwin, H.L. Ball, N. Bradon, et al., J. Mol. Biol. 314 (2001) 1209–1225. [51] A.D. Frankel, C.O. Pabo, Cell 55 (1988) 1189–1193. [52] M. Green, P.M. Loewenstein, Cell 55 (1988) 1179–1188. [53] E. Vives, P. Brodin, B. Lebleu, J. Biol. Chem. 272 (1997) 16010–16017. [54] D. Derossi, A.H. Joliot, G. Chassaing, A. Prochiantz, J. Biol. Chem. 269 (1994) 10444–10450. [55] S. Console, C. Marty, C. Garcia-Echeverria, R. Schwendener, K. Ballmer-Hofer, J. Biol. Chem. 278 (2003) 35109–35114. [56] F. Duchardt, M. Fotin-Mleczek, H. Schwarz, R. Fischer, R. Brock, Traffic 8 (2007) 848–866. [57] M. Magzoub, A. Pramanik, A. Gräslund, Biochemistry 44 (2005) 14890–14897. [58] E. Ghibaudi, B. Boscolo, G. Inserra, E. Laurenti, S. Traversa, et al., J. Pept. Sci. 11 (2005) 401–409. [59] P. Deriziotis, R. Andre, D.M. Smith, R. Goold, K.J. Kinghorn, et al., EMBO J. 30 (2011) 3065–3077. [60] A. Rigter, J.P. Langeveld, F.G. van Zijderveld, A. Bossers, Vaccine 28 (2010) 7810–7823. [61] I.H. Solomon, N. Khatri, E. Biasini, T. Massignan, J.E. Huettner, et al., J. Biol. Chem. 286 (2011) 14724–14736. [62] J.A. Turnbaugh, U. Unterberger, P. Saa, T. Massignan, B.R. Fluharty, et al., J. Neurosci. 32 (2012) 8817–8830. [63] V.G. Ostapchenko, N. Makarava, R. Savtchenko, I.V. Baskakov, J. Mol. Biol. 383 (2008) 1210–1224. [64] F. Madani, R. Abdo, S. Lindberg, H. Hirose, S. Futaki, et al., Biochim. Biophys. Acta 1828 (2013) 1198–1204. [65] K. Kapp, S. Schrempf, M.K. Lemberg, B. Dobberstein, in: R. Zimmermann (Ed.), Protein Transport into the Endoplasmic Reticulum, Austin, TX, Landes Bioscience, 2009, pp. 1–16. [66] D.I. Chan, E.J. Prenner, H.J. Vogel, in: Biochim. Biophys. Acta 1758 (2006) 1184– 1202. [67] M.C. Kerr, R.D. Teasdale, Traffic 10 (2009) 364–371. [68] N.D. Younan, C.J. Sarell, P. Davies, D.R. Brown, J.H. Viles, FASEB J. (2013). [69] L.O. Tjernberg, J. Näslund, F. Lindqvist, J. Johansson, A.R. Karlström, et al., J. Biol. Chem. 271 (1996) 8545–8548. [70] M.H. Viet, S.T. Ngo, N.S. Lam, M.S. Li, J. Phys. Chem. B 115 (2011) 7433–7446. [71] D.R. Taylor, I.J. Whitehouse, N.M. Hooper, PLoS Pathog. 5 (2009) e1000666. [72] H. Kubota, J. Biochem. 146 (2009) 609–616. [73] P.J. Peters, A. Mironov Jr., D. Peretz, E. van Donselaar, E. Leclerc, et al., J. Cell Biol. 162 (2003) 703–717. [74] M. Vey, S. Pilkuhn, H. Wille, R. Nixon, S.J. DeArmond, et al., Proc. Natl. Acad. Sci. U.S.A. 93 (1996) 14945–14949.