Chirality Switching within an Anionic Cell-Penetrating Peptide Inhibits

Nov 25, 2014 - Division of Surgical Oncology, Department of Surgery, University of Illinois College of ... uptake of anionic peptides into cancer cell...
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Chirality Switching within an Anionic Cell-Penetrating Peptide Inhibits Translocation without Affecting Preferential Entry Tohru Yamada,† Sara Signorelli,‡ Salvatore Cannistraro,‡ Craig W. Beattie,† and Anna Rita Bizzarri*,‡ †

Division of Surgical Oncology, Department of Surgery, University of Illinois College of Medicine, Chicago, Illinois 60612, United States ‡ Biophysics and Nanoscience Centre, CNISM-DEB, Università della Tuscia, Viterbo, Italy ABSTRACT: Multiple substitution of D- for L-amino acids decreases the intracellular uptake of cationic cell penetrating peptides (CPP) in a cell line-dependent manner. We show here that a single D-amino acid substitution can decrease the overall uptake of the anionic, amphipathic CPP, p28, into cancer and histologically matched normal cell lines, while not altering the preferential uptake of p28 into cancer cells. The decrease appears dependent on the position of the D-substitution within the peptide and the ability of the substituted D-amino acid to alter chirality. We also suggest that when D-substitution alters the ratio of α-helix to β-sheet content of an anionic CPP, its translocation across the cell membrane is altered, reducing overall entry. These observations may have a significant effect on the design of future D-substituted analogues of cell penetrating peptides. KEYWORDS: cell penetrating peptides, chirality, Raman spectroscopy, circular dichroism, intracellular uptake

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INTRODUCTION Cell penetrating peptides (CPP) are generally described as short peptides of less than 30 amino acids that possess a positive net charge and are able to penetrate biological membranes via receptor mediated endocytotic pathways as well as direct translocation.1 Recently, anionic, amphipathic CPPs that enter cells by endocytotic and nonendocytotic (energy independent) mechanisms2 and induce active proteins have been reported.3−9 Despite a relatively simple structural organization, anionic CPPs share a set of molecular features similar to cationic CPPs. These include amphipathicity and spatial distribution of charge and hydrophobic/hydrophilic properties. However, the mechanisms of anionic peptide endocytotic entry and intracellular transport are significantly different from those generally accepted for cationic or positively charged CPPs.2,10 Although the energy independent (direct translocation) uptake mechanism used by anionic CPPs is, as yet, unknown in detail, like cationic CPPs, physical−chemical interactions between the peptide and lipid bilayers likely govern peptide translocation independent of receptor mediated processes.11−13 Membrane-associated folding of a CPP is one potential source of energy for moving through the hydrophobic layer of a membrane bilayer, but does not completely explain translocation at 4 °C.2 It also does not explain the preferential uptake of anionic peptides into cancer cells relative to histologically normal cells at similar temperatures.2 The role of peptide secondary structure in cell penetration also remains elusive. As a peptide’s secondary structure depends on its environment,14 peptides can adopt different conforma© 2014 American Chemical Society

tions depending on whether they are in water, near the membrane interface, inside the membrane, or bound to a protein. Secondary structure within a peptide class (e.g., cationic, amphipathic, or hydrophobic) also appears to influence the mode of uptake.14 Although linear, cationic αhelical peptides represent the most studied class of CPPs,15 CPPs apparently adopt either a β-sheet conformation or mix of α-helical and β-sheet structures over a broad range of peptide concentrations depending on the lipid composition of a particular membrane contact site.16 This suggests that α-helical and β-sheet structures underlie, at least in part, CPP translocation through cell membranes, although this concept remains arguable.11 Moreover, initial reports that amphipathic, α-helical17 and β-sheet CPPs18 are also sensitive to mutations and alterations in chirality that disrupt their 3D structure and significantly reduce uptake have recently been confirmed for amphipathic and nonamphipathic, cationic CPPs.19 p28, amino acids (aa) 50 to 77 of azurin, a cupredoxin secreted by Pseudomonas aeruginosa, is a unique amphipathic, anionic CPP predicted to comprise α-helical and β-sheet motifs within its parent protein. It preferentially enters cancer and developing endothelial cells via a defined endocytotic pathway and by direct translocation.2,3,5,7 The mechanism underlying the latter pathway remains undefined. Although molecular Received: Revised: Accepted: Published: 140

July 21, 2014 October 29, 2014 November 25, 2014 November 25, 2014 dx.doi.org/10.1021/mp500495u | Mol. Pharmaceutics 2015, 12, 140−149

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at 37 °C for 2 h in prewarmed medium containing Alexa Fluor 568-labeled peptides at 20 μM or medium alone. After incubation, coverslips were washed, fixed, and mounted in medium containing 1.5 μg/mL 4′,6-diamidino-2-phenylindole (DAPI) to counterstain nuclei (VECTASHIELD; Vector Laboratories, Burlingame, CA). Cellular uptake and distribution of peptides were photographed under an inverted confocal laser scanning microscope (LC510 and 710 META; Carl Zeiss Inc.).2 Cell Penetration by Fluorescence Activated Cell Sorting (FACS). FACS analysis was performed essentially as described.2 Cells were incubated for 2 h at 37 °C with Alexa Fluor 568-labeled peptides (20 μM) or medium alone, washed in PBS, fixed in 2.5% formalin, resuspended in PBS, and analyzed with a MoFlo Cell Sorter (Dako, Carpinteria, CA) and LSRFortessa (BD Biosciences, San Jose, CA). The fold increase of the mean fluorescence intensity (MFI) over background levels represents mean fluorescence of three separate experiments. Entry inhibitor: cells were pretreated with 5 mM of methyl-β-cyclodextrin (MβCD) (Sigma-Aldrich), inhibitor of caveolae-mediated endocytosis. Final concentration was derived from the dose response curve of the inhibitor.2 The kinetics of p28 entry2 at 4 °C were established in UISO-Mel-2 cells suspended in MEME media without phenol red at 5 × 105 cells/tube. Reactions were initiated by adding 400 μM Alexa Fluor 568-conjugated p28 for 30−120 s on ice. After incubation, 1 mL of cold-PBS was added to the 250 μL reaction in mixture. Cells were washed extensively with PBS and centrifuged twice at 600 × g for 2 min at 4 °C. Background and relative fluorescence were analyzed in 10,000 fixed cells by flow cytometry in each reaction, and Km and Vmax calculated as concentration vs velocity (MFI/seconds). Raman Measurements. Raman spectra were recorded using a Jobin-Yvon Super Labram confocal system equipped with a liquid nitrogen-cooled CCD (EEV CCD10-11 back illuminated; pixel format, 1024 × 128) detector and a spectrograph with a 1800 g/mm grating allowing a resolution of 5 cm−1. The laser source was Argon ion laser (MellesGriot) providing a 514.5 nm radiation with a power kept below 12 mW (corresponding to about 60 kW/cm2); the real power impinging on the sample being below 1.9 mW. Raman spectra of each peptide (3 mM) were collected in the backscattering geometry and a notch filter was used to reject the elastic contribution, thus preventing also the collection of spectra close to the excitation line. A 50× objective with a numerical aperture NA = 0.6 was implemented and a large confocal diaphragm of 600 μm has been used in order to obtain a good Raman signal. The typical acquisition time was 5 min. All the spectra were fitted using Levenberg−Marquardt minimization algorithm (LMA) and Lorentzian−Gaussian pseudo-Voigt functions as peak profiles. Circular Dichroism (CD). CD measurements were carried out on a JASCO J-710 Spectropolarimeter (Easton, MD). Optical cell of 1 mm path length was placed in a thermostable cell holder. CD spectra were collected on 100 μM of peptide solutions in 100% PBS, 100% TFE, or methanol/H2O (50%/ 50%) by running two scans at 1 nm intervals from 190−260 nm at 298 K. Secondary structures (% helix and % β-sheet) of each peptide were estimated with K2D3 software.23

dynamic simulations15 and solid state NMR20,21 provide an entrée into predicting how a cationic CPP translocates cell membranes, the lack of positive charge and lipid solubility, respectively, of anionic peptides limits their applicability. Here we use classical CD coupled with Raman spectrometry, confocal microscopy, and flow cytometry to describe the transition states of p28 (a model anionic CPP) and show how changes in secondary structure and chirality alter the overall, but not the preferential uptake of CPPs into cancer and normal cells.

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EXPERIMENTAL SECTION Peptide Synthesis. All azurin-derived peptides including p18, p28, and D-substituted p28 analogues were synthesized by C S Bio, Inc. (Menlo Park, CA), at >95% purity and mass balance. p28 is an amphipathic peptide (2.9 kDa) and aa residues Leu 50 -Asp 77 of Azurin (LSTAADMQGVVTDGMASGLDKDYLKPDD). p18 is an α-helical peptide (1.7 kDa) aa residues Leu50-Gly67 of azurin (LSTAADMQGVVTDGMASG). Chirality was altered at dL1-p28, dD22-p28, dL24p28, and dD28-p28, where d indicates D-isomer substitution at L (leucine) or D (aspartic acid) amino acid. Sample Preparation. All the samples were dissolved in three different solvents: 100% PBS (phosphate buffered saline, 95.3% H2O, 3.8% NaCl, 0.1% di KCl, 0.7% Na2HPO4, and 0.1% KH2PO4, pH = 7.5); 100% TFE (trifluoroethanol, CF3CH2OH; Sigma-Aldrich, St. Louis, MO); and 50% methanol (CH3OH) and 50% H2O. Cell Culture. Human cancer and noncancer (immortalized and nonimmortalized) cell lines were obtained from American Type Culture Collection (Manassas, VA): lung cancer (A549, H69AR, and NCI-H23 adenocarcinoma); normal lung (CCD13Lu); kidney cancer (CRL-1611); normal kidney (HK-2); liver cancer (HepG2); normal liver (THLE-2); prostate cancers (DU145, LNCaP, and PC-3); normal prostate (CRL11611); breast cancer (MCF-7, MDA-MB-231, T47D, and ZR-75-1); normal breast (MCF-10A); colon cancer (BE, Colo205, HCT116, HT29, SW620, and W1Dr); normal colon (CCD33Co); astrocytoma (CCF-STTG1); glioblastoma (U87 and LN229); neuroblastoma (IMR-32 and SK-N-BE); fibrosarcoma (HT1080); rhabdomyosarcoma (RD); osteosarcoma (TE85); leiomyosarcoma (HTB88); and ovarian cancer (ES-2, CAOV-3, SKOV3, and PA-1). Bladder cancer (BLD-1), breast cancer (BCA1 and BCA2), colon cancer (CCa9 and CCa12), Ewing sarcoma (ES3), giant cell tumor (UISO-GCT1), prostate cancer (UISO-PR-1), and normal fibroblasts isolated from skin were established in our laboratory. Breast cancer (MDD2) and normal ovarian cells (HOSE6-3) were a generous gift from Dr. Andrei V. Gudkov (Roswell Park Cancer Institute) and Dr. S. W. Tsao (University of Hong Kong, Hong Kong, China), respectively. Melanoma lines (UISO-Mel-2, -6, -23, and -29) were established and characterized in our laboratory.22 U87, normal kidney, and normal live cells were maintained in Dullbecco’s MEM, Keratinocyte-SFM (Invitrogen, Carlsbad, CA), and EBM-2 medium (Lonza Inc., Walkersville, MD), respectively. All other cell lines except UISO-Mel-2 (MEM-H) were cultured in MEM-E (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum (Atlanta Biological, Inc.), 100 units/mL penicillin, and 100 μg/ mL streptomycin at 37 °C in 5% CO2 or air. Confocal Microscopy. p28 and its analogues were labeled with Alexa Fluor 568 dye (Life Technologies, NY) as described.2 Cells were seeded on glass coverslips, incubated

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RESULTS Cellular Uptake of p28. p28 preferentially penetrates cancer cells (Figure 1) with overall entry into histologically 141

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Figure 1. Preferential penetration of p28 into human cancer cells. FACS analyses (Summit ver. 4.3) of p28 entry into human cancer (n = 44) and histologically matched normal cell lines. Values represent calculated fold increase over fluorescence from normal cells. Preferential (entry above red line); >95%, ≥2-fold increase; ∼71% over normal cells of the same histologic subtype. Mean ± SE of ≥3 independent observations.

matched normal and cancer cells directly attributable to endocytosis within and between cell types (Figure 2). Although the degree of inhibition of caveolae-mediated endocytosis is directly related to the amount of p28 that enters endocytotically (Figure 2A), MβCD did not completely prevent entry into either cancer (∼50−60%) or normal cells (∼40−50%) (Figure 2B), an observation similar to that observed for filipin and nystatin, additional inhibitors of caveolae mediated endocytosis.2 Although cell penetration and intracellular transport of p28 is temperature dependent (Figure 2C), entry kinetics into UISO-Mel-2 cells at 4 °C is also time- and dose-related (Figure 2D) and apparently involves more than endocytosis or ATP.2 Unlike cationic CPPs, p28 does not remain at the cell membrane, even at 4 °C (Figure 2C), again suggesting its mode of entry and defined intracellular transport2 result, in part, from lipid raft-mediated and clathrin- and caveolinindependent nonendocytosis pathways24 and not the uptake related fixation artifact common to cationic CPPs.25 Direct translocation appears to provide a significant avenue for p28 entry into cancer and normal cells,2 possibly based on the lipid composition of membrane contact sites. The predicted structure 26 of p28 within its parent protein, Azurin, incorporates a sequential series of α-helical and β-sheet motifs (Figure 3A). As an amphipathic peptide, its hydrophilic and hydrophobic amino acids are distributed throughout each predicted helix and the single β-sheet (Figure 3B). However, the first 18 amino acids comprise the more hydrophobic part of the peptide, while the c-terminal 10 is essentially hydrophilic amino acids including K21and K25 (Figure 3C). This distribution is reflected in the lack of preferential entry of the terminal 12 amino acids of p28, a peptide that readily enters all cells to a similar degree.2 Membrane-associated peptide folding is one potential source of energy for moving through the hydrophobic region of a membrane bilayer, but does not completely explain the diminished but still significant translocation of p28 at 4 °C.2 As the folding process is environment-dependent, we studied the potential role of an increasing secondary structure on cell penetration by sequentially analyzing the behavior of native unfolded, anionic p28 (theoretical pI 3.9) in phosphate saline buffer, progressing through mixed (methanol/water) and nonaqueous environments (trifluoroethanol) by CD and

Figure 2. Endocytotic and nonendocytotic penetration of p28. (A) Linear regression of p28 entry in the presence or absence of methyl-βcyclodextrin (MβCD) was determined by FACS. Human cancer and normal cell lines were pretreated with 5 mM MβCD for 60 min and incubated with 20 μM Alexa Fluor 568-labeled p28. Intracellular fluorescence intensity in the absence of MβCD (control) was considered as 100%. (B) Ratio of endocytotic and nonendocytotic penetration of p28 in melanoma (Mel2), fibroblast, ovarian (cancer SKOV3 and normal HOSE 6-3), and prostate (cancer DU145) and normal prostate cells (CRL11611) in the presence of 5 mM MβCD. Nonendocytotic entry is the difference between total entry expressed as 100% and that inhibited by MβCD. Mean ± SE of ≥3 independent observations. (C) Temperature-dependent penetration of p28 into UISO-Mel-2 cells. Penetration of Alexa Fluor 568-labeled p28 at 20 μM for 2 h at 4, 8, 22, and 37 °C was evaluated by confocal microscopy. Red, Alexa Fluor-labeled p28; blue, DAPI (nucleus). (D) Kinetics of p28 entry. UISO-Mel-2 cells were suspended in MEME, and reactions were started by adding Alexa Fluor 568-conjugated p28 at 10, 50, 100, 150, 250, 300, and 400 μM for 30, 60, 90, and 120 s on ice. The Km and Vmax values of p28 were calculated by plotting p28 concentration (μM) vs velocity (MFI/sec).

Raman spectroscopy.27 An amide I Raman band, which arises from the sum of coupled modes of the polypeptide backbone,28 is strongly dependent on backbone secondary structure and specially sensitive to changes in its conformation in the way it reflects vibrational couplings and hydrogen bonding due to the nature (polarity) of the solvent environment.29 The addition of methanol, which promotes a β-sheet formation, allowed us to assess the relative flexibility of p2830 and the potential contribution a β-sheet structure could have on penetration. CD Analysis of Secondary Structure. An initial statistical analysis of the evolution of p28 secondary structure in PBS, over multiple molecular dynamics simulations,8,31 predicted that p28 forms α-helical (35%) and β-sheet (21%) motifs with the remaining peptide a random coil. Table 1 describes the CD 142

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was complete, where it decreased to essentially zero (Table 1), suggesting its absence within a lipid membrane. The ratio of αhelix (TFE)/β-sheet (MeOH/H2O) was ∼6.0, reflecting the increase in α-helical content. Single D-amino acid substitutions in either the n- or c-terminal α-helix had varying effects on the secondary structure of p28 in MeOH/H2O and TFE. dL1-p28 did not alter either the increase in α-helix content or the αhelix/β-sheet ratio (Table 1), while dL22, dL24, and dD28 significantly reduced the amount of α-helix in MeOH/H2O and TFE (dL24) and the α-helix/β-sheet ratio of all three peptides (Table 1). This suggested that alterations in chirality that reduced α-helical secondary structure were dependent on the location of those substitutions and potentially significant on overall cell penetration. Raman Spectroscopic Analysis of Secondary Structure. We complemented our CD analyses with Raman spectroscopy and spectral fitting to extract information about the vibrational frequencies and the relaxation rates associated with random coil to α-helix conformational transitions. This assured all α-helices were identified and completely characterized.27 Raman spectroscopy of the amide I group in the three solvents provided the basis for analyzing p28 in its native or unfolded state as modified by an increasing organic phase as well as alterations in chirality. The Raman spectra in the region of 400−1800 cm−1 of p28 in PBS, MeOH/H2O (1:1) solution, and TFE are shown in Figure 4A. The five peaks at the frequencies 646, 830, 853, 1177, and 1615 cm−1 arise from the aromatic ring of the unique amino acid Tyr in p28’s sequence with C−C stretching, an amide III band, and CH2−CH3 deformation observed at 950, 1252, and 1424 cm−1 , respectively. The region 1650−1700 cm−1 was characterized by the vibration mode of the amide I, that arises mainly from the CO stretching vibration with minor contributions from the out-of-phase C−N stretching vibration, the C−C−N deformation, and the N−H in-plane bend.32 The spectra of p28 in the three solvents exhibited similar peaks with small shifts and slight variations in both intensity and width of the line, depending on the polarity of the solvents (Figure 4A). In particular, Tyr was relatively insensitive to the change from a polar (PBS) to less polar (MeOH) or nonpolar (TFE) solvent. In contrast, the amide I band was influenced by a change in solvent polarity, with the frequency of the central peak shifting from 1666 in PBS to 1671 cm−1 in TFE. The width at half height (fwhm) was decreased from ∼50 cm−1 in PBS to ∼45 and 40 cm−1 in MeOH/H2O and TFE, respectively. Lowering

Figure 3. Structural characteristics of p28. (A) Robson plot for p28. αHelical motif (blue) and β-sheet (green). Primary sequence of p28 is shown above the Robson plot. Positions of D-substitution are also indicated (arrow). (B) Helical wheel representation of p28. DSubstitutions (O). Hydrophilic (red), hydrophobic (blue), and neutral (black) residues. (C) Kyte-Doolittle (55) hydropathy plot of p28. Scores above and below the line indicate the degree of hydrophobicity and hydrophilicity, respectively. Amino acid sequence of p28 is indicated below the plot. GENETYX ver 6.1 was used for all analyses.

spectra for p28 and D-substituted analogues in PBS, MeOH/ H2O, and TFE. CD provided an initial global estimate of the role of secondary structure on penetration of a cell’s membrane. Positions of D-substituted amino acids are indicated in Figure 3A,B. In PBS, p28 was essentially unfolded with minimal αhelix (6%) and β-sheet (11%) somewhat lower than predicted by the model. Exposure to MeOH/H2O doubled its α-helical content (11%), which rose to 69% in TFE. There was no significant alteration in β-sheet content until the organic phase Table 1. Circular Dichroism of p28 and Its Analoguesa PBS p28 dL1 dD22 dL24 dD28

% % % % % % % % % %

helix β-sheet helix β-sheet helix β-sheet helix β-sheet helix β-sheet

MeOH/H2O

TFE

mean

SD

mean

SD

mean

SD

ratio (% helix in TFE/% β-sheet in MeOH/H2O)

6.1 11.1 5.0 11.9 4.0 13.3 4.6 14.2 4.1 11.8

1.4 0.2 0.1 0.2 0.1 0.1 0.2 0.6 0.1 0.1

11.4 11.3 10.3 10.9 5.7 12.9 2.3 13.9 5.8 12.3

1.1 0.2 0.5 0.1 0.1 0.2 0.7 0.2 0.1 0.4

69.2 0.2 67.0 0.1 70.7 0.2 55.1 1.4 67.4 0.1

0.1 0.1 0.2 0.1 0.8 0.1 0.1 0.1 0.1 0.1

6.1 6.1 5.5 4.0 5.5

CD spectra (190−260 nm) were obtained on p28 and D-substituted analogues in PBS, MeOH/H2O (50%/50%), and TFE at 25 °C. Secondary structures of each peptide were estimated with the K2D3 server (http://k2d3.ogic.ca/).

a

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Figure 4. Raman spectra of p28 and its analogues. (A) The Raman spectra of p28 at 514.5 nm, 23 °C, were obtained over 400−1800 cm−1 in PBS (black), MeOH/H2O (1:1) (blue), and TFE (red). Major band frequencies are indicated (1650−1700 cm−1 for the amide I band, 646, 830, 853, 1177, and 1615 cm−1 for Tyr, and 950, 1252, and 1424 cm−1 for C−C stretching, amide III band, and CH2−CH3 deformation, respectively). (B) The amide I peak of p28 (-o-, red) at 1650−1700 cm−1 in PBS (left panel), MeOH/H2O (middle panel), and TFE (right panel). Deconvolution revealed the relative contribution of each secondary structure, α-helix, β-sheet, and RC (random coil), 2nd, 3rd, and 4th peak from left (green). The band at 1615 cm−1 (1st peak from left) was included in the curve-fitting protocol to account for ring modes from aromatic Tyr residue. Vibrational frequency (cm−1) of each peak is indicated. (C) Raman spectra (600−1800 cm−1) of p28, dL1, dD22, dL24, and dD28 at 514.5 nm in PBS, MeOH/ H2O, and TFE. Representative Raman spectra of p28 and its analogues in PBS (left panel, (a)) and spectra of dL1 in PBS (black), MeOH/H2O (blue), and TFE (red) (right panel, (b)) are illustrated. (D) The relative contributions (% area) of each secondary structure, α-helix, β-sheet, and RC, obtained from the amide I band of p28 and D-substituted p28 analogues in PBS, MeOH/H2O, and TFE were calculated from the integrated area of each principal component. The helix/β-sheet ratio (%) in TFE and MeOH/H2O is also shown.

solvent polarity also resulted in a narrower peak, suggesting dominance of one particular configuration in secondary

structure. As the Raman amide I band was broad, it suggested a mixture of conformations.30 Further analyses,33 including 144

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Figure 5. Penetration of p28 and D-substituted analogues into matched cancer and normal cell lines. Cells plated on coverslips were incubated with 20 μM of Alexa Fluor 568-labeled p28 or its analogues at 37 °C for 2 h, and images were recorded by confocal microscopy. Arrowheads indicate the localization of p28. All L-p28, dL1, dL24, and dD28 localized in the endoplasmic reticulum (ER) and cell nucleus. dD22 localized essentially within the cytoplasm. Red, p28/analogues; blue, DAPI (nucleus); green, auto fluorescence.

intramolecular configuration. Further analysis of the Raman spectra also suggest that alterations in chirality appear to reduce the amide I and III bonds in p28, in PBS (Figure 4C (a)). Although each solvent induced similar small shifts and slight variations in both the line intensity and width with p28 and dL1-p28, the amide I region was clearly increased in the latter as the organic phase is increased (Figure 4C (b)). The general increase in β-sheet content in MeOH/H2O, not observed with CD, is not sustained in TFE where the α-helical content increases significantly (Figure 4D). Here again, the MeOH/ H2O/TFE α-helix/β-sheet ratio is significantly altered by the location of individual D-substitutions (Figure 4D). Preferential Uptake of p28 and D-Substituted Analogues. We determined whether each alteration in the α-helix/ β-sheet ratio predicted by CD and Raman spectrometry was reflected in overall and preferential cell penetration with confocal microscopy (Figure 5) and flow cytometry (Figure 6) using three histologically matched cancer and normal cell lines expressing different degrees of preferential penetration (Figure 1). Except for dL1-p28, the penetration of dD22, dL24, and dD28-p28 was less than that of p28, reflecting the altered αhelix/β-sheet ratio demonstrated by CD. Although entry was reduced, dL24 and dD28-p28 were present in the cytoplasm, the ER, and nucleus, a distribution similar to p28 (Figure 5). The singular exception was dD22-p28. Here, uptake was significantly reduced with minimal uptake from the cytoplasm into the ER and nucleus relative to p28 (Figure 5). This

spectral decomposition of the amide I band in terms of its different components, provided the relative contribution from α-helices, β-sheets, and random coils.34 When band intensity was integrated with the corresponding amide I peak, the Raman cross section of each conformation was identical.30 A simple curve-fitting technique was applied to dissect the contribution of different secondary conformations (Figure 4B).35 Curve fitting of the p28 Raman spectra in three solvents revealed that three bands were sufficient to reproduce the amide I feature (Figure 4B). The relative contribution of each secondary structure, e.g., α-helix, was calculated from the integrated area of each principal component (Figure 4D). We included the integrated area of the band at 1615 cm−1 in the band-fitting protocol to account for ring modes from the aromatic Tyr residue,36 but did not report it in Table 1. The major component was at about 1680 cm−1 in PBS (45% random coil). In sum, Raman results show that p28 is highly flexible and modified by its local environment. A random coil dominates in PBS, in good agreement with the model,31,37 but unlike results with CD, there was a significant increase in β-sheet content induced by MeOH/H2O. The α-helical content of p28 was further increased in the presence of TFE. The Raman spectra of D-substituted p28 in PBS and the spectra of dL1-p28 in the three solvents are shown in Figure 4C (a) and (b), respectively. The peak ascribed to the Tyr at about 642 cm−1 was minimal in the spectra from dL1-p28, dD22, dL24, and dD28 (Figure 4C (a)) indicating a different 145

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DISCUSSION

The physical basis for membrane translocation of CPPs is not well understood.39 Virtually all amphipathic and nonamphipathic cationic CPPs bind to heparin sulfate (HS) on a cell membrane and are subsequently internalized endocytotically.14,40 At higher protein concentrations a net positive charge increases their solubility in lipid vesicles that model aspects of the cell membrane.41 For example, ion pair interaction between the Arg residues of nonamphipathic cationic peptides, such as HIV1 TAT and penetratin, and anionic lipid head groups may play a significant role in initiating the membrane translocation of such CPPs.21,42 The significant random coil and lack of amphipathic structure inherent in these peptides has also been suggested to be essential for the rapid translocation of this type of CPP across the lipid membrane without causing its disruption.21 However, penetratin and other cationic peptides also reportedly adopt α-helical43 or βsheet13,42 structures in addition to appearing as a random coil when contacting anionic lipids. They can also exhibit a dual behavior by presenting either their cationic or hydrophobic domains toward the phospholipid face depending on the nature of the lipid (anionic or zwitterionic).43 Overall, the interaction of cationic CPPs with the membrane clearly involves an initial electrostatic interaction with cell surface proteoglycans that triggers a conformational transition of the peptide from random coil to β-sheet or α-helical forms readily observeable by CD.13 This mechanism is not available to anionic CPPs as amphipathic, anionic CPPs are insoluble in neutral (cholesterol) and negatively charged POPC (1-palmitoyl-2-oleoyl-snglycero-3-phosphocholine)/cholesterol (3:2), POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine)/POPG (1palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol), POPC/ POPG, POPC/POPS (1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine)/cholesterol, and POPA (1-palmitoyl-2-oleoyl-snglycero-3-phosphate) lipids adjusted for packing density, even at high molar ratios (1:4) makes either CD or Raman spectroscopic analysis as well as solid state NMR difficult.42 Preferential accumulation of selected cationic CPPs in cancer cell membranes at sites that overexpress certain proteoglycans rich in anionic components44,45 also does not appear relevant for anionic CPPs. Amphipathic, cationic CPPs are also subject to cell type specific uptake mechanisms40 that appear dependent on alterations in chirality, particularly those that affect an α-helical backbone.18,19 Incorporation of D-amino acids into CPPs decreases their potential, proteolytic instability,46 a major limitation in the pharmacological use of CPPs,47 but also alters chirality. Altering the chirality of linear, cationic CPPs reportedly results in a reduction in internalization, but requires prior removal of the HS chains that bind cationic CPPs to the cell membrane.19 This is not required for amphipathic, anionic CPPs, such as p28, as they do not bind to HS.2 To date, studies on the role of chirality in affecting cell entry have not addressed either the role of translocation relative to endocytosis during internalization or the preferential penetration of amphipathic, anionic CPPs into cancer and developing endothelial cells.2,7 We address those issues here and suggest that the ratio of αhelix/β-sheet motifs evolves as an anionic CPP transitions through the cell membrane facilitating its translocation. We show that the position of D-amino acid substitutions within a αhelical backbone, and possibly the strength of the substitutions that alter chirality,38 may be critical to both the endocytosis and

Figure 6. Flow cytometric analysis of p28 and D-substituted analogue penetration. (A) Cells were incubated with 20 μM of Alexa Fluor 568labeled p28 or its analogues at 37 °C for 2 h and mean fluorescence intensities determined by FACS. (B) Fold increase in fluorescence of p28 and D-substituted analogues over histologically matched normal cells. Mean ± SE of 3 independent observations.

suggests that alterations in chirality may also affect intracellular distribution. The increase in the α-helix/β-sheet ratio demonstrated by Raman spectroscopy for dD22 and dL24p28 mirrored the alterations observed with CD, with the exception of dD28-p28 where the ratio was similar to that of p28 and dL1-p28. This suggests that the position of the Dsubstitution within the α-helix may be critical to overall uptake. When position is coupled with a weak helix destabilizer (DAsp), 38 it suggests the notable increase in C−C−N deformation may compensate for the loss in the amide I peak (Figure 4C (a)) to affect penetration. Alternatively, an increase in α-helical content in the transitional environment of MeOH/ H2O and an α-helix/β-sheet ratio close to p28 may predict an uptake (Table 1; Figure 4D) similar to p28. Flow cytometric analyses of the entry of D-substituted p28 analogues confirmed and enhanced these observations (Figure 6). The overall penetration of dL1-p28 was not significantly different to that of p28, irrespective of cell line, while that of dL24 and dD28 were uniformly lower (Figure 6A). The entry of dD22-p28 was, again, significantly lower than that of p28 or the other Danalogues (Figure 6A). In sharp contrast, preferential penetration of all D-substituted analogues of p28 into cancer cells (fold increase over matched normal cells) was essentially identical to that of 28 (Figure 6B). This suggests that discrete changes in chirality alter the overall translocation of p28, but not the preferential penetration of amphipathic, anionic CPPs into cancer cells. The significant positional effect of single D-substituton of p28 on overall penetration and intracellular distribution also negates any increase in overall uptake and intracellular redistribution reportedly induced by cell fixation prior to the analysis of the uptake of highly cationic CPPs.25 When coupled with the lack of membrane binding (Figures 2C and 6) and significant reduction in the penetration of labeled p28 in the presence of unlabeled p28 in fixed cells,2 it further suggests that the penetration and intracellular distribution of p28 is the result of a saturable and specific process and not an artifact of the fixation process.24 146

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μM, the concentration used here, of cationic CPPs enter the cytoplasm directly52 through spatially confined areas of the plasma membrane via an acid sphingomyelinase-dependent mechanism that changes the lipid composition of the plasma membrane but maintains plasma membrane integrity.51 The insolubility of anionic p28 in anionic lipids suggests this may not be the route by which p28 translocates across a cell membrane. However, anionic CPPs, like cationic CPPs, clearly adopt different conformational states depending on their environment. They exist in a disordered or random coil form in water and adopt a α-helical structure in organic solvents.14,31,37 As such, secondary structure appears as the single constant in the entry of this unique anionic CPP and amphipathic cationic CPPs. p28, like its amphipathic cationic counterparts, enters from a high dielectric medium (water) to a low dielectric medium (core of the membrane). This suggests that the hydrophilic, cationic c-terminal 12 (p12) and hydrophobic nterminal 16−18 amino acids (Figure 3B,C) distributed along the second α-helix and initial α-helix and β-sheet (Figure 3A), respectively, combined with an average reduction in the charge, may act in concert to, first, introduce the peptide to the cell membrane and, second, assist in crossing the core of the cell membrane through hydrophobic interaction.53 The initial increase in α-helix and maintenance of the β-sheet content (Table 1) observed as solvent polarity is reduced, and significant increase in α-helicity and loss of β-sheet as it is reduced further also suggest that secondary structure may be critical for the translocation, if not the endocytosis of anionic CPPs.2 Although not quite as extensive in overall and preferential entry into cancer cells, a similar behavior is observed for amino acids 50 to 67 of p28 (p18) the initial hydrophobic α-helix and β-sheet of p28 and minimal motif (protein transduction domain) responsible for the preferential entry of Azurin into human cancer cells.2 Although the net charge of p28, p18, and p12 are all negative (−4, −2, and −2, respectively), the overall hydrophobic scores54 of p28, p18, and p12 are −0.35, 0.34, and −1.3, respectively. This suggests that a reduction in hydrophobicity is reflected in the lack of preferential entry of p12.2 The reduction in overall and preferential entry of p18, relative to p28, may also result from the lack of a hydrophilic, cationic α-helix reducing its entry pathway to that through endocytosis.2 Irrespective of route of entry, CD and Raman spectroscopy appear to be useful tools for predicting the entry of amphipathic, anionic peptides. There was a uniform increase in the α-helical content of all L-p28 in TFE with both approaches, suggesting that as p28 approaches the cell membrane its structure changes from largely random coil to α-helix while maintaining a relative stable degree of β-sheet, which decreases to essentially zero as an α-helical secondary structure penetrates the bilipid membrane and traverses the cytoplasm, late endosomes, and ER to the nucleus.2 The significant decrease and increase in the α-helix/β-sheet ratio of p28 and D-substituted analogues, observed with CD and Raman spectroscopy, respectively, provides a certain degree of predictability to the overall entry of a D-amino acid substituted anionic CPP. Figure 2A,B shows that endocytotic penetration into cancer cell lines is significantly higher than matched normal cells with significant entry (40−60%) occurring in both via a nonendocytotic pathway. This suggests that the degree of endocytosis affects not only overall penetration but also the

translocation of amphipathic, anionic CPPs. Finally, we also show that chirality can reduce the overall entry of a CPP, but not the preferential nature of its penetration. A uniform increase in α-helix content of p28 and all Dsubstituted analogues in TFE and virtual lack of β-sheet relative to that in PBS and MeOH/H2O (Table 1 and Figure 4D) confirms the necessity of an α-helical motif for entry of anionic as well as cationic CPPs.43,48 It also predicts that a reduction in β-sheet content may also be essential for CPP penetration without membrane disruption, a hallmark of β-sheet poreforming CPPs. As a class, these latter peptides undergo a spontaneous transition in which loop sequences change conformation and are inserted into a cell or bacterial membrane to form β-barrel pores.49 This may explain why amphipathic anionic CPPs do not form pores nor disrupt a cell membrane during entry.2 p28 and its D-analogues showed a stable β-sheet content in PBS and MeOH/H2O by CD analysis. This provided a platform to examine the ratio of β-sheet to α-helix content (TFE) within the context of a lipid membrane. All Dsubstitutions within the c-terminal α-helix reduced this ratio (Table 1), which predicted a decrease in cell entry. This was reflected in the Raman analyses of each analogue in MeOH/ H2O, which saw a significant increase in β-sheet content from that in PBS and α-helix in TFE, the latter in close agreement with results from CD. However, the α-helix/β-sheet ratio for p28 was the reverse from that seen with CD as was that from dL1-p28 and dD28-p28. The α-helix/β-sheet ratio for dD22and dL24-p28 was greater than unity and predictive of the decrease in overall penetration of these analogues. The lack of correlation between the α-helix/β-sheet ratio observed with CD and Raman spectra and overall entry of dD28-p28 suggests that either the position of the D-substitution within this α-helix or weakness in strength of this substitution to alter chirality may be critical to translocation. As D-amino acids cause only a local change in structure and flexibility at the position of the substitution in an α-helical backbone,50 the strength of the substitutions that alter chirality38 at selected positions may also be critical to the endocytosis and translocation of amphipathic, anionic CPPs. The turn-like structures induced by all D-amino acids to destabilize α-helices are reportedly highly dependent on the amino acid side chain and not related to the structure propensity of the corresponding L-amino acid.38 The possibility that position along the α-helical backbone may be critical is strengthened by our observation that D-Asp (dD22-p28) is a weak helix destabilizer, dL24-p28 is a medium destabilizer, and dD28-p28 is, again, weak. The latter two substitutions reduce penetration (Figures 5 and 6), but not as significantly as dD22p28, making it reasonable to predict that location and D-amino acid influence cell penetration via either endocytosis or translocation. Finally, we also show that altering chirality can reduce the overall entry of an amphipathic, anionic CPP (Figures 5 and 6A), but does not alter the preferential nature of its penetration (Figure 6B). This finding can be broadly applied to the overall uptake of cationic CPPs for at least two reasons. First, partial Dsubstitution of cationic, nonstructured, nonamphipathic CPPs also appears to reduce their uptake by endocytosis in a cell type-dependent manner. Second, a consecutive stretch of Lamino acids is required to trigger uptake.19,51 Our observation that a single D-substitution along an α-helical backbone can affect overall uptake is comparable. Although uptake appeared to be restricted to endocytosis, higher concentrations, e.g., 20 147

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p53 without altering its conformation. Mol. Pharmaceutics 2013, 10 (9), 3375−3383. (7) Mehta, R. R.; Yamada, T.; Taylor, B. N.; Christov, K.; King, M. L.; Majumdar, D.; Lekmine, F.; Tiruppathi, C.; Shilkaitis, A.; Bratescu, L.; Green, A.; Beattie, C. W.; Das Gupta, T. K. A cell penetrating peptide derived from azurin inhibits angiogenesis and tumor growth by inhibiting phosphorylation of VEGFR-2, FAK and Akt. Angiogenesis 2011, 14, 355−369. (8) Bizzarri, A. R.; Santini, S.; Coppari, E.; Bucciantini, M.; Di Agostino, S.; Yamada, T.; Beattie, C. W.; Cannistraro, S. Interaction of an anticancer peptide fragment of azurin with p53 and its isolated domains studied by atomic force spectroscopy. Int. J. Nanomed. 2011, 6, 3011−3019. (9) Yamada, T.; Christov, K.; Shilkaitis, A.; Bratescu, L.; Green, A.; Santini, S.; Bizzarri, A. R.; Cannistraro, S.; Gupta, T. K.; Beattie, C. W. p28, A first in class peptide inhibitor of cop1 binding to p53. Br. J. Cancer. 2013, 108, 2495−2504. (10) Bechara, C.; Sagan, S. Cell-penetrating peptides: 20 years later, where do we stand? FEBS Lett. 2013, 587, 1693−1702. (11) Derossi, D.; Calvet, S.; Trembleau, A.; Brunissen, A.; Chassaing, G.; Prochiantz, A. Cell internalization of the third helix of the Antennapedia homeodomain is receptor-independent. J. Biol. Chem. 1996, 271, 18188−18193. (12) Deshayes, S.; Konate, K.; Aldrian, G.; Crombez, L.; Heitz, F.; Divita, G. Structural polymorphism of non-covalent peptide-based delivery systems: highway to cellular uptake. Biochim. Biophys. Acta 2010, 1798, 2304−2314. (13) Eiriksdottir, E.; Konate, K.; Langel, U.; Divita, G.; Deshayes, S. Secondary structure of cell-penetrating peptides controls membrane interaction and insertion. Biochim. Biophys. Acta 2010, 1798, 1119− 1128. (14) Milletti, F. Cell-penetrating peptides: classes, origin, and current landscape. Drug Discovery Today 2012, 17, 850−860. (15) Polyansky, A. A.; Chugunov, A. O.; Vassilevski, A. A.; Grishin, E. V.; Efremov, R. G. Recent advances in computational modeling of alpha-helical membrane-active peptides. Curr. Protein Pept. Sci. 2012, 13, 644−657. (16) Ding, B.; Chen, Z. Molecular interactions between cell penetrating peptide Pep-1 and model cell membranes. J. Phys. Chem. B 2012, 116, 2545−2552. (17) Oehlke, J.; Scheller, A.; Wiesner, B.; Krause, E.; Beyermann, M.; Klauschenz, E.; Melzig, M.; Bienert, M. Cellular uptake of an alphahelical amphipathic model peptide with the potential to deliver polar compounds into the cell interior non-endocytically. Biochim. Biophys. Acta 1998, 1414, 127−139. (18) Oehlke, J.; Krause, E.; Wiesner, B.; Beyermann, M.; Bienert, M. Extensive cellular uptake into endothelial cells of an amphipathic betasheet forming peptide. FEBS Lett. 1997, 415, 196−199. (19) Verdurmen, W. P.; Bovee-Geurts, P. H.; Wadhwani, P.; Ulrich, A. S.; Hallbrink, M.; van Kuppevelt, T. H.; Brock, R. Preferential uptake of L- versus D-amino acid cell-penetrating peptides in a cell type-dependent manner. Chem. Biol. 2011, 18, 1000−1010. (20) Su, Y.; Li, S.; Hong, M. Cationic membrane peptides: atomiclevel insight of structure-activity relationships from solid-state NMR. Amino Acids 2013, 44, 821−833. (21) Su, Y.; Waring, A. J.; Ruchala, P.; Hong, M. Membrane-bound dynamic structure of an arginine-rich cell-penetrating peptide, the protein transduction domain of HIV TAT, from solid-state NMR. Biochemistry 2010, 49, 6009−6020. (22) Rauth, S.; Kichina, J.; Green, A.; Bratescu, L.; Das Gupta, T. K. Establishment of a human melanoma cell line lacking p53 expression and spontaneously metastasizing in nude mice. Anticancer Res. 1994, 14, 2457−2463. (23) Louis-Jeune, C.; Andrade-Navarro, M. A.; Perez-Iratxeta, C. Prediction of protein secondary structure from circular dichroism using theoretically derived spectra. Proteins 2011, 80, 374−381. (24) Rhee, M.; Davis, P. Mechanism of uptake of C105Y, a novel cellpenetrating peptide. J. Biol. Chem. 2006, 281, 1233−1240.

preferential penetration of p28 into cancer cells (Figure 1). p28 is an amphipathic α-helical peptide with a stretch of hydrophobic amino acids predicted to be clustered about the n-terminal α-helix and β-sheet.14 D-substitution of the nterminal leucine of p28 does not alter the α-helix/β-sheet ratio (Table 1 and Figure 4D), overall entry, or intracellular distribution (Figure 5), confirming that altering the chirality of the initial amino acid in the n-terminal α-helix does not alter either endocytotic or direct entry into cells. The single substitutions within the c-terminal at positions 22, 24, and 28 of the p28 α-helix reduced the overall entry of these analogues into all cell types irrespective of the degree of overall entry. In sharp contrast, preferential penetration of all D-substituted analogues of p28 into cancer cells (fold increase over matched normal cells) was essentially identical to that of p28 (Figure 6B). This suggests that discrete changes in chirality can alter overall entry, but not the preferential penetration of amphipathic, anionic CPPs. In summary, although details for the mechanism that p28 and other anionic CPPs2 use to translocate a cell membrane remain unknown, it appears that the ratio of α-helix/β-sheet motifs evolves as an anionic CPP transitions through the cell membrane facilitating its translocation. The position of D-amino acid substitutions within the α-helical backbone and the strength of the substitutions that alter chirality38 may also be critical to the overall entry of a CPP, but not the preferential nature of its penetration.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +39 0761 357031. Fax: +39 0761 357027. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was partly supported by a grant from the Italian Association for Cancer Research (AIRC No IG 10412) and by a PRIN-MIUR 2012 Project (No. 2012NRRP5J).

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REFERENCES

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