chirality switching within an anionic cell-penetrating...

Chirality Switching within an Anionic Cell-Penetrating PeptideInhibits Translocation without Affecting Preferential EntryTohru 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, UnitedStates‡Biophysics and Nanoscience Centre, CNISM-DEB, Universita della Tuscia, Viterbo, Italy

ABSTRACT: Multiple substitution of D- for L-amino acids decreasesthe intracellular uptake of cationic cell penetrating peptides (CPP) ina cell line-dependent manner. We show here that a single D-aminoacid substitution can decrease the overall uptake of the anionic,amphipathic CPP, p28, into cancer and histologically matchednormal cell lines, while not altering the preferential uptake of p28into cancer cells. The decrease appears dependent on the position ofthe D-substitution within the peptide and the ability of thesubstituted D-amino acid to alter chirality. We also suggest thatwhen D-substitution alters the ratio of α-helix to β-sheet content ofan anionic CPP, its translocation across the cell membrane is altered,reducing overall entry. These observations may have a significanteffect on the design of future D-substituted analogues of cellpenetrating peptides.

KEYWORDS: cell penetrating peptides, chirality, Raman spectroscopy, circular dichroism, intracellular uptake

■ INTRODUCTION

Cell penetrating peptides (CPP) are generally described asshort peptides of less than 30 amino acids that possess apositive net charge and are able to penetrate biologicalmembranes via receptor mediated endocytotic pathways aswell as direct translocation.1 Recently, anionic, amphipathicCPPs that enter cells by endocytotic and nonendocytotic(energy independent) mechanisms2 and induce active proteinshave been reported.3−9 Despite a relatively simple structuralorganization, anionic CPPs share a set of molecular featuressimilar to cationic CPPs. These include amphipathicity andspatial distribution of charge and hydrophobic/hydrophilicproperties. However, the mechanisms of anionic peptideendocytotic entry and intracellular transport are significantlydifferent from those generally accepted for cationic or positivelycharged CPPs.2,10 Although the energy independent (directtranslocation) uptake mechanism used by anionic CPPs is, asyet, unknown in detail, like cationic CPPs, physical−chemicalinteractions between the peptide and lipid bilayers likely governpeptide translocation independent of receptor mediatedprocesses.11−13 Membrane-associated folding of a CPP is onepotential source of energy for moving through the hydrophobiclayer of a membrane bilayer, but does not completely explaintranslocation at 4 °C.2 It also does not explain the preferentialuptake of anionic peptides into cancer cells relative tohistologically normal cells at similar temperatures.2

The role of peptide secondary structure in cell penetrationalso remains elusive. As a peptide’s secondary structure dependson its environment,14 peptides can adopt different conforma-

tions depending on whether they are in water, near themembrane interface, inside the membrane, or bound to aprotein. Secondary structure within a peptide class (e.g.,cationic, amphipathic, or hydrophobic) also appears toinfluence the mode of uptake.14 Although linear, cationic α-helical peptides represent the most studied class of CPPs,15

CPPs apparently adopt either a β-sheet conformation or mix ofα-helical and β-sheet structures over a broad range of peptideconcentrations depending on the lipid composition of aparticular membrane contact site.16 This suggests that α-helicaland β-sheet structures underlie, at least in part, CPPtranslocation through cell membranes, although this conceptremains arguable.11 Moreover, initial reports that amphipathic,α-helical17 and β-sheet CPPs18 are also sensitive to mutationsand alterations in chirality that disrupt their 3D structure andsignificantly reduce uptake have recently been confirmed foramphipathic and nonamphipathic, cationic CPPs.19

p28, amino acids (aa) 50 to 77 of azurin, a cupredoxinsecreted by Pseudomonas aeruginosa, is a unique amphipathic,anionic CPP predicted to comprise α-helical and β-sheet motifswithin its parent protein. It preferentially enters cancer anddeveloping endothelial cells via a defined endocytotic pathwayand by direct translocation.2,3,5,7 The mechanism underlyingthe latter pathway remains undefined. Although molecular

Received: July 21, 2014Revised: October 29, 2014Accepted: November 25, 2014Published: November 25, 2014

Article

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© 2014 American Chemical Society 140 dx.doi.org/10.1021/mp500495u | Mol. Pharmaceutics 2015, 12, 140−149

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dynamic simulations15 and solid state NMR20,21 provide anentree into predicting how a cationic CPP translocates cellmembranes, the lack of positive charge and lipid solubility,respectively, of anionic peptides limits their applicability. Herewe use classical CD coupled with Raman spectrometry,confocal microscopy, and flow cytometry to describe thetransition states of p28 (a model anionic CPP) and show howchanges in secondary structure and chirality alter the overall,but not the preferential uptake of CPPs into cancer and normalcells.

■ EXPERIMENTAL SECTIONPeptide Synthesis. All azurin-derived peptides including

p18, p28, and D-substituted p28 analogues were synthesized byC S Bio, Inc. (Menlo Park, CA), at >95% purity and massbalance. p28 is an amphipathic peptide (2.9 kDa) and aaresidues Leu50-Asp77of Azurin (LSTAADMQGVVTDG-MASGLDKDYLKPDD). p18 is an α-helical peptide (1.7kDa) aa residues Leu50-Gly67 of azurin (LSTAADMQGVVT-DGMASG). Chirality was altered at dL1-p28, dD22-p28, dL24-p28, and dD28-p28, where d indicates D-isomer substitution atL (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 AmericanType Culture Collection (Manassas, VA): lung cancer (A549,H69AR, and NCI-H23 adenocarcinoma); normal lung (CCD-13Lu); 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); osteosar-coma (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 andCCa12), Ewing sarcoma (ES3), giant cell tumor (UISO-GCT-1), prostate cancer (UISO-PR-1), and normal fibroblastsisolated from skin were established in our laboratory. Breastcancer (MDD2) and normal ovarian cells (HOSE6-3) were agenerous gift from Dr. Andrei V. Gudkov (Roswell Park CancerInstitute) and Dr. S. W. Tsao (University of Hong Kong, HongKong, China), respectively. Melanoma lines (UISO-Mel-2, -6,-23, and -29) were established and characterized in ourlaboratory.22 U87, normal kidney, and normal live cells weremaintained in Dullbecco’s MEM, Keratinocyte-SFM (Invitro-gen, Carlsbad, CA), and EBM-2 medium (Lonza Inc.,Walkersville, MD), respectively. All other cell lines exceptUISO-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) asdescribed.2 Cells were seeded on glass coverslips, incubated

at 37 °C for 2 h in prewarmed medium containing Alexa Fluor568-labeled peptides at 20 μM or medium alone. Afterincubation, coverslips were washed, fixed, and mounted inmedium containing 1.5 μg/mL 4′,6-diamidino-2-phenylindole(DAPI) to counterstain nuclei (VECTASHIELD; VectorLaboratories, Burlingame, CA). Cellular uptake and distributionof peptides were photographed under an inverted confocal laserscanning microscope (LC510 and 710 META; Carl ZeissInc.).2

Cell Penetration by Fluorescence Activated CellSorting (FACS). FACS analysis was performed essentially asdescribed.2 Cells were incubated for 2 h at 37 °C with AlexaFluor 568-labeled peptides (20 μM) or medium alone, washedin PBS, fixed in 2.5% formalin, resuspended in PBS, andanalyzed with a MoFlo Cell Sorter (Dako, Carpinteria, CA) andLSRFortessa (BD Biosciences, San Jose, CA). The fold increaseof the mean fluorescence intensity (MFI) over backgroundlevels represents mean fluorescence of three separate experi-ments. Entry inhibitor: cells were pretreated with 5 mM ofmethyl-β-cyclodextrin (MβCD) (Sigma-Aldrich), inhibitor ofcaveolae-mediated endocytosis. Final concentration was derivedfrom the dose response curve of the inhibitor.2 The kinetics ofp28 entry2 at 4 °C were established in UISO-Mel-2 cellssuspended in MEME media without phenol red at 5 × 105

cells/tube. Reactions were initiated by adding 400 μM AlexaFluor 568-conjugated p28 for 30−120 s on ice. Afterincubation, 1 mL of cold-PBS was added to the 250 μLreaction in mixture. Cells were washed extensively with PBSand centrifuged twice at 600 × g for 2 min at 4 °C. Backgroundand relative fluorescence were analyzed in 10,000 fixed cells byflow cytometry in each reaction, and Km and Vmax calculated asconcentration vs velocity (MFI/seconds).

Raman Measurements. Raman spectra were recordedusing a Jobin-Yvon Super Labram confocal system equippedwith a liquid nitrogen-cooled CCD (EEV CCD10-11 backilluminated; pixel format, 1024 × 128) detector and aspectrograph with a 1800 g/mm grating allowing a resolutionof 5 cm−1. The laser source was Argon ion laser (MellesGriot)providing a 514.5 nm radiation with a power kept below 12mW (corresponding to about 60 kW/cm2); the real powerimpinging on the sample being below 1.9 mW. Raman spectraof each peptide (3 mM) were collected in the backscatteringgeometry and a notch filter was used to reject the elasticcontribution, thus preventing also the collection of spectra closeto the excitation line. A 50× objective with a numerical apertureNA = 0.6 was implemented and a large confocal diaphragm of600 μm has been used in order to obtain a good Raman signal.The typical acquisition time was 5 min. All the spectra werefitted using Levenberg−Marquardt minimization algorithm(LMA) and Lorentzian−Gaussian pseudo-Voigt functions aspeak profiles.

Circular Dichroism (CD). CD measurements were carriedout on a JASCO J-710 Spectropolarimeter (Easton, MD).Optical cell of 1 mm path length was placed in a thermostablecell holder. CD spectra were collected on 100 μM of peptidesolutions in 100% PBS, 100% TFE, or methanol/H2O (50%/50%) by running two scans at 1 nm intervals from 190−260 nmat 298 K. Secondary structures (% helix and % β-sheet) of eachpeptide were estimated with K2D3 software.23

■ RESULTSCellular Uptake of p28. p28 preferentially penetrates

cancer cells (Figure 1) with overall entry into histologically

Molecular Pharmaceutics Article

dx.doi.org/10.1021/mp500495u | Mol. Pharmaceutics 2015, 12, 140−149141

matched normal and cancer cells directly attributable toendocytosis within and between cell types (Figure 2). Althoughthe degree of inhibition of caveolae-mediated endocytosis isdirectly related to the amount of p28 that enters endocytotically(Figure 2A), MβCD did not completely prevent entry intoeither cancer (∼50−60%) or normal cells (∼40−50%) (Figure2B), an observation similar to that observed for filipin andnystatin, additional inhibitors of caveolae mediated endocy-tosis.2 Although cell penetration and intracellular transport ofp28 is temperature dependent (Figure 2C), entry kinetics intoUISO-Mel-2 cells at 4 °C is also time- and dose-related (Figure2D) and apparently involves more than endocytosis or ATP.2

Unlike cationic CPPs, p28 does not remain at the cellmembrane, even at 4 °C (Figure 2C), again suggesting itsmode of entry and defined intracellular transport2 result, inpart, from lipid raft-mediated and clathrin- and caveolin-independent nonendocytosis pathways24 and not the uptakerelated fixation artifact common to cationic CPPs.25 Directtranslocation appears to provide a significant avenue for p28entry into cancer and normal cells,2 possibly based on the lipidcomposition of membrane contact sites. The predictedstructure26 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 andhydrophobic amino acids are distributed throughout eachpredicted helix and the single β-sheet (Figure 3B). However,the first 18 amino acids comprise the more hydrophobic part ofthe peptide, while the c-terminal 10 is essentially hydrophilicamino acids including K21and K25 (Figure 3C). Thisdistribution is reflected in the lack of preferential entry of theterminal 12 amino acids of p28, a peptide that readily enters allcells to a similar degree.2

Membrane-associated peptide folding is one potential sourceof energy for moving through the hydrophobic region of amembrane bilayer, but does not completely explain thediminished but still significant translocation of p28 at 4 °C.2

As the folding process is environment-dependent, we studiedthe potential role of an increasing secondary structure on cellpenetration by sequentially analyzing the behavior of nativeunfolded, anionic p28 (theoretical pI 3.9) in phosphate salinebuffer, progressing through mixed (methanol/water) andnonaqueous environments (trifluoroethanol) by CD and

Raman spectroscopy.27 An amide I Raman band, which arisesfrom the sum of coupled modes of the polypeptide backbone,28

is strongly dependent on backbone secondary structure andspecially sensitive to changes in its conformation in the way itreflects vibrational couplings and hydrogen bonding due to thenature (polarity) of the solvent environment.29 The addition ofmethanol, which promotes a β-sheet formation, allowed us toassess the relative flexibility of p2830 and the potentialcontribution a β-sheet structure could have on penetration.

CD Analysis of Secondary Structure. An initial statisticalanalysis of the evolution of p28 secondary structure in PBS,over multiple molecular dynamics simulations,8,31 predictedthat p28 forms α-helical (35%) and β-sheet (21%) motifs withthe remaining peptide a random coil. Table 1 describes the CD

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 representcalculated 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 ≥3independent observations.

Figure 2. Endocytotic and nonendocytotic penetration of p28. (A)Linear regression of p28 entry in the presence or absence of methyl-β-cyclodextrin (MβCD) was determined by FACS. Human cancer andnormal cell lines were pretreated with 5 mM MβCD for 60 min andincubated with 20 μM Alexa Fluor 568-labeled p28. Intracellularfluorescence intensity in the absence of MβCD (control) wasconsidered as 100%. (B) Ratio of endocytotic and nonendocytoticpenetration of p28 in melanoma (Mel2), fibroblast, ovarian (cancerSKOV3 and normal HOSE 6-3), and prostate (cancer DU145) andnormal prostate cells (CRL11611) in the presence of 5 mM MβCD.Nonendocytotic entry is the difference between total entry expressedas 100% and that inhibited by MβCD. Mean ± SE of ≥3 independentobservations. (C) Temperature-dependent penetration of p28 intoUISO-Mel-2 cells. Penetration of Alexa Fluor 568-labeled p28 at 20μM for 2 h at 4, 8, 22, and 37 °C was evaluated by confocalmicroscopy. 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 p28at 10, 50, 100, 150, 250, 300, and 400 μM for 30, 60, 90, and 120 s onice. The Km and Vmax values of p28 were calculated by plotting p28concentration (μM) vs velocity (MFI/sec).



spectra for p28 and D-substituted analogues in PBS, MeOH/H2O, and TFE. CD provided an initial global estimate of therole of secondary structure on penetration of a cell’s membrane.Positions of D-substituted amino acids are indicated in Figure3A,B. In PBS, p28 was essentially unfolded with minimal α-helix (6%) and β-sheet (11%) somewhat lower than predictedby the model. Exposure to MeOH/H2O doubled its α-helicalcontent (11%), which rose to 69% in TFE. There was nosignificant alteration in β-sheet content until the organic phase

was complete, where it decreased to essentially zero (Table 1),suggesting its absence within a lipid membrane. The ratio of α-helix (TFE)/β-sheet (MeOH/H2O) was ∼6.0, reflecting theincrease in α-helical content. Single D-amino acid substitutionsin either the n- or c-terminal α-helix had varying effects on thesecondary structure of p28 in MeOH/H2O and TFE. dL1-p28did not alter either the increase in α-helix content or the α-helix/β-sheet ratio (Table 1), while dL22, dL24, and dD28significantly reduced the amount of α-helix in MeOH/H2O andTFE (dL24) and the α-helix/β-sheet ratio of all three peptides(Table 1). This suggested that alterations in chirality thatreduced α-helical secondary structure were dependent on thelocation of those substitutions and potentially significant onoverall cell penetration.

Raman Spectroscopic Analysis of Secondary Struc-ture. We complemented our CD analyses with Ramanspectroscopy and spectral fitting to extract information aboutthe vibrational frequencies and the relaxation rates associatedwith random coil to α-helix conformational transitions. Thisassured all α-helices were identified and completely charac-terized.27 Raman spectroscopy of the amide I group in the threesolvents provided the basis for analyzing p28 in its native orunfolded state as modified by an increasing organic phase aswell as alterations in chirality. The Raman spectra in the regionof 400−1800 cm−1 of p28 in PBS, MeOH/H2O (1:1) solution,and TFE are shown in Figure 4A. The five peaks at thefrequencies 646, 830, 853, 1177, and 1615 cm−1 arise from thearomatic ring of the unique amino acid Tyr in p28’s sequencewith C−C stretching, an amide III band, and CH2−CH3deformation observed at 950, 1252, and 1424 cm−1,respectively. The region 1650−1700 cm−1 was characterizedby the vibration mode of the amide I, that arises mainly fromthe CO stretching vibration with minor contributions fromthe out-of-phase C−N stretching vibration, the C−C−Ndeformation, and the N−H in-plane bend.32 The spectra ofp28 in the three solvents exhibited similar peaks with smallshifts and slight variations in both intensity and width of theline, depending on the polarity of the solvents (Figure 4A). Inparticular, Tyr was relatively insensitive to the change from apolar (PBS) to less polar (MeOH) or nonpolar (TFE) solvent.In contrast, the amide I band was influenced by a change insolvent polarity, with the frequency of the central peak shiftingfrom 1666 in PBS to 1671 cm−1 in TFE. The width at halfheight (fwhm) was decreased from ∼50 cm−1 in PBS to ∼45and 40 cm−1 in MeOH/H2O and TFE, respectively. Lowering

Figure 3. Structural characteristics of p28. (A) Robson plot for p28. α-Helical motif (blue) and β-sheet (green). Primary sequence of p28 isshown above the Robson plot. Positions of D-substitution are alsoindicated (arrow). (B) Helical wheel representation of p28. D-Substitutions (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 hydrophobicityand hydrophilicity, respectively. Amino acid sequence of p28 isindicated below the plot. GENETYX ver 6.1 was used for all analyses.

Table 1. Circular Dichroism of p28 and Its Analoguesa

PBS MeOH/H2O TFE

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

p28 % helix 6.1 1.4 11.4 1.1 69.2 0.1 6.1% β-sheet 11.1 0.2 11.3 0.2 0.2 0.1

dL1 % helix 5.0 0.1 10.3 0.5 67.0 0.2 6.1% β-sheet 11.9 0.2 10.9 0.1 0.1 0.1

dD22 % helix 4.0 0.1 5.7 0.1 70.7 0.8 5.5% β-sheet 13.3 0.1 12.9 0.2 0.2 0.1

dL24 % helix 4.6 0.2 2.3 0.7 55.1 0.1 4.0% β-sheet 14.2 0.6 13.9 0.2 1.4 0.1

dD28 % helix 4.1 0.1 5.8 0.1 67.4 0.1 5.5% β-sheet 11.8 0.1 12.3 0.4 0.1 0.1

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



http://k2d3.ogic.ca/

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

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

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). Deconvolutionrevealed the relative contribution of each secondary structure, α-helix, β-sheet, and RC (random coil), 2nd, 3rd, and 4th peak from left (green). Theband at 1615 cm−1 (1st peak from left) was included in the curve-fitting protocol to account for ring modes from aromatic Tyr residue. Vibrationalfrequency (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, andRC, obtained from the amide I band of p28 and D-substituted p28 analogues in PBS, MeOH/H2O, and TFE were calculated from the integrated areaof each principal component. The helix/β-sheet ratio (%) in TFE and MeOH/H2O is also shown.



spectral decomposition of the amide I band in terms of itsdifferent components, provided the relative contribution fromα-helices, β-sheets, and random coils.34 When band intensitywas integrated with the corresponding amide I peak, the Ramancross section of each conformation was identical.30 A simplecurve-fitting technique was applied to dissect the contributionof different secondary conformations (Figure 4B).35 Curvefitting of the p28 Raman spectra in three solvents revealed thatthree bands were sufficient to reproduce the amide I feature(Figure 4B). The relative contribution of each secondarystructure, e.g., α-helix, was calculated from the integrated areaof each principal component (Figure 4D). We included theintegrated area of the band at 1615 cm−1 in the band-fittingprotocol to account for ring modes from the aromatic Tyrresidue,36 but did not report it in Table 1. The majorcomponent was at about 1680 cm−1 in PBS (45% random coil).In sum, Raman results show that p28 is highly flexible andmodified by its local environment. A random coil dominates inPBS, in good agreement with the model,31,37 but unlike resultswith CD, there was a significant increase in β-sheet contentinduced by MeOH/H2O. The α-helical content of p28 wasfurther 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 about642 cm−1 was minimal in the spectra from dL1-p28, dD22,dL24, and dD28 (Figure 4C (a)) indicating a different

intramolecular configuration. Further analysis of the Ramanspectra also suggest that alterations in chirality appear to reducethe amide I and III bonds in p28, in PBS (Figure 4C (a)).Although each solvent induced similar small shifts and slightvariations in both the line intensity and width with p28 anddL1-p28, the amide I region was clearly increased in the latteras the organic phase is increased (Figure 4C (b)). The generalincrease in β-sheet content in MeOH/H2O, not observed withCD, is not sustained in TFE where the α-helical contentincreases significantly (Figure 4D). Here again, the MeOH/H2O/TFE α-helix/β-sheet ratio is significantly altered by thelocation of individual D-substitutions (Figure 4D).

Preferential Uptake of p28 and D-Substituted Ana-logues. We determined whether each alteration in the α-helix/β-sheet ratio predicted by CD and Raman spectrometry wasreflected in overall and preferential cell penetration withconfocal microscopy (Figure 5) and flow cytometry (Figure 6)using three histologically matched cancer and normal cell linesexpressing different degrees of preferential penetration (Figure1). Except for dL1-p28, the penetration of dD22, dL24, anddD28-p28 was less than that of p28, reflecting the altered α-helix/β-sheet ratio demonstrated by CD. Although entry wasreduced, 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 wassignificantly reduced with minimal uptake from the cytoplasminto the ER and nucleus relative to p28 (Figure 5). This

Figure 5. Penetration of p28 and D-substituted analogues into matched cancer and normal cell lines. Cells plated on coverslips were incubated with20 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 thelocalization of p28. All L-p28, dL1, dL24, and dD28 localized in the endoplasmic reticulum (ER) and cell nucleus. dD22 localized essentially withinthe cytoplasm. Red, p28/analogues; blue, DAPI (nucleus); green, auto fluorescence.



suggests that alterations in chirality may also affect intracellulardistribution. The increase in the α-helix/β-sheet ratiodemonstrated by Raman spectroscopy for dD22 and dL24-p28 mirrored the alterations observed with CD, with theexception of dD28-p28 where the ratio was similar to that ofp28 and dL1-p28. This suggests that the position of the D-substitution within the α-helix may be critical to overall uptake.When position is coupled with a weak helix destabilizer (D-Asp),38 it suggests the notable increase in C−C−Ndeformation may compensate for the loss in the amide I peak(Figure 4C (a)) to affect penetration. Alternatively, an increasein α-helical content in the transitional environment of MeOH/H2O and an α-helix/β-sheet ratio close to p28 may predict anuptake (Table 1; Figure 4D) similar to p28. Flow cytometricanalyses of the entry of D-substituted p28 analogues confirmedand enhanced these observations (Figure 6). The overallpenetration of dL1-p28 was not significantly different to that ofp28, irrespective of cell line, while that of dL24 and dD28 wereuniformly lower (Figure 6A). The entry of dD22-p28 was,again, significantly lower than that of p28 or the other D-analogues (Figure 6A). In sharp contrast, preferentialpenetration of all D-substituted analogues of p28 into cancercells (fold increase over matched normal cells) was essentiallyidentical to that of 28 (Figure 6B). This suggests that discretechanges in chirality alter the overall translocation of p28, butnot the preferential penetration of amphipathic, anionic CPPsinto cancer cells.The significant positional effect of single D-substituton of p28

on overall penetration and intracellular distribution also negatesany increase in overall uptake and intracellular redistributionreportedly induced by cell fixation prior to the analysis of theuptake of highly cationic CPPs.25 When coupled with the lackof membrane binding (Figures 2C and 6) and significantreduction in the penetration of labeled p28 in the presence ofunlabeled p28 in fixed cells,2 it further suggests that thepenetration and intracellular distribution of p28 is the result ofa saturable and specific process and not an artifact of thefixation process.24

■ DISCUSSION

The physical basis for membrane translocation of CPPs is notwell understood.39 Virtually all amphipathic and nonamphi-pathic cationic CPPs bind to heparin sulfate (HS) on a cellmembrane and are subsequently internalized endocytoti-cally.14,40 At higher protein concentrations a net positivecharge increases their solubility in lipid vesicles that modelaspects of the cell membrane.41 For example, ion pairinteraction between the Arg residues of nonamphipathiccationic peptides, such as HIV1 TAT and penetratin, andanionic lipid head groups may play a significant role in initiatingthe membrane translocation of such CPPs.21,42 The significantrandom coil and lack of amphipathic structure inherent in thesepeptides has also been suggested to be essential for the rapidtranslocation of this type of CPP across the lipid membranewithout causing its disruption.21 However, penetratin and othercationic peptides also reportedly adopt α-helical43 or β-sheet13,42 structures in addition to appearing as a random coilwhen contacting anionic lipids. They can also exhibit a dualbehavior by presenting either their cationic or hydrophobicdomains toward the phospholipid face depending on the natureof the lipid (anionic or zwitterionic).43 Overall, the interactionof cationic CPPs with the membrane clearly involves an initialelectrostatic interaction with cell surface proteoglycans thattriggers a conformational transition of the peptide from randomcoil to β-sheet or α-helical forms readily observeable by CD.13

This mechanism is not available to anionic CPPs asamphipathic, anionic CPPs are insoluble in neutral (cholester-ol) and negatively charged POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine)/cholesterol (3:2), POPE (1-palmi-toyl-2-oleoyl-sn-glycero-3-phosphoethanolamine)/POPG (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol), POPC/POPG, POPC/POPS (1-palmitoyl-2-oleoyl-sn-glycero-3-phos-pho-L-serine)/cholesterol, and POPA (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate) lipids adjusted for packing density, evenat high molar ratios (1:4) makes either CD or Ramanspectroscopic analysis as well as solid state NMR difficult.42

Preferential accumulation of selected cationic CPPs in cancercell membranes at sites that overexpress certain proteoglycansrich in anionic components44,45 also does not appear relevantfor anionic CPPs.Amphipathic, cationic CPPs are also subject to cell type

specific uptake mechanisms40 that appear dependent onalterations in chirality, particularly those that affect an α-helicalbackbone.18,19 Incorporation of D-amino acids into CPPsdecreases their potential, proteolytic instability,46 a majorlimitation in the pharmacological use of CPPs,47 but also alterschirality. Altering the chirality of linear, cationic CPPsreportedly results in a reduction in internalization, but requiresprior removal of the HS chains that bind cationic CPPs to thecell membrane.19 This is not required for amphipathic, anionicCPPs, such as p28, as they do not bind to HS.2 To date, studieson the role of chirality in affecting cell entry have not addressedeither the role of translocation relative to endocytosis duringinternalization 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 α-helix/β-sheet motifs evolves as an anionic CPP transitionsthrough the cell membrane facilitating its translocation. Weshow that the position of D-amino acid substitutions within a α-helical backbone, and possibly the strength of the substitutionsthat alter chirality,38 may be critical to both the endocytosis and

Figure 6. Flow cytometric analysis of p28 and D-substituted analoguepenetration. (A) Cells were incubated with 20 μM of Alexa Fluor 568-labeled p28 or its analogues at 37 °C for 2 h and mean fluorescenceintensities determined by FACS. (B) Fold increase in fluorescence ofp28 and D-substituted analogues over histologically matched normalcells. Mean ± SE of 3 independent observations.



translocation of amphipathic, anionic CPPs. Finally, we alsoshow that chirality can reduce the overall entry of a CPP, butnot the preferential nature of its penetration.A uniform increase in α-helix content of p28 and all D-

substituted analogues in TFE and virtual lack of β-sheet relativeto that in PBS and MeOH/H2O (Table 1 and Figure 4D)confirms the necessity of an α-helical motif for entry of anionicas well as cationic CPPs.43,48 It also predicts that a reduction inβ-sheet content may also be essential for CPP penetrationwithout membrane disruption, a hallmark of β-sheet pore-forming CPPs. As a class, these latter peptides undergo aspontaneous transition in which loop sequences changeconformation and are inserted into a cell or bacterial membraneto form β-barrel pores.49 This may explain why amphipathicanionic CPPs do not form pores nor disrupt a cell membraneduring entry.2

p28 and its D-analogues showed a stable β-sheet content inPBS and MeOH/H2O by CD analysis. This provided aplatform to examine the ratio of β-sheet to α-helix content(TFE) within the context of a lipid membrane. All D-substitutions within the c-terminal α-helix reduced this ratio(Table 1), which predicted a decrease in cell entry. This wasreflected in the Raman analyses of each analogue in MeOH/H2O, which saw a significant increase in β-sheet content fromthat in PBS and α-helix in TFE, the latter in close agreementwith results from CD. However, the α-helix/β-sheet ratio forp28 was the reverse from that seen with CD as was that fromdL1-p28 and dD28-p28. The α-helix/β-sheet ratio for dD22-and dL24-p28 was greater than unity and predictive of thedecrease in overall penetration of these analogues. The lack ofcorrelation between the α-helix/β-sheet ratio observed with CDand Raman spectra and overall entry of dD28-p28 suggests thateither the position of the D-substitution within this α-helix orweakness in strength of this substitution to alter chirality maybe critical to translocation. As D-amino acids cause only a localchange in structure and flexibility at the position of thesubstitution in an α-helical backbone,50 the strength of thesubstitutions that alter chirality38 at selected positions may alsobe critical to the endocytosis and translocation of amphipathic,anionic CPPs. The turn-like structures induced by all D-aminoacids to destabilize α-helices are reportedly highly dependenton the amino acid side chain and not related to the structurepropensity of the corresponding L-amino acid.38 The possibilitythat position along the α-helical backbone may be critical isstrengthened by our observation that D-Asp (dD22-p28) is aweak helix destabilizer, dL24-p28 is a medium destabilizer, anddD28-p28 is, again, weak. The latter two substitutions reducepenetration (Figures 5 and 6), but not as significantly as dD22-p28, making it reasonable to predict that location and D-aminoacid influence cell penetration via either endocytosis ortranslocation.Finally, we also show that altering chirality can reduce the

overall entry of an amphipathic, anionic CPP (Figures 5 and6A), but does not alter the preferential nature of its penetration(Figure 6B). This finding can be broadly applied to the overalluptake of cationic CPPs for at least two reasons. First, partial D-substitution of cationic, nonstructured, nonamphipathic CPPsalso appears to reduce their uptake by endocytosis in a celltype-dependent manner. Second, a consecutive stretch of L-amino acids is required to trigger uptake.19,51 Our observationthat a single D-substitution along an α-helical backbone canaffect overall uptake is comparable. Although uptake appearedto be restricted to endocytosis, higher concentrations, e.g., 20

μM, the concentration used here, of cationic CPPs enter thecytoplasm directly52 through spatially confined areas of theplasma membrane via an acid sphingomyelinase-dependentmechanism that changes the lipid composition of the plasmamembrane but maintains plasma membrane integrity.51 Theinsolubility of anionic p28 in anionic lipids suggests this maynot be the route by which p28 translocates across a cellmembrane.However, anionic CPPs, like cationic CPPs, clearly adopt

different conformational states depending on their environ-ment. They exist in a disordered or random coil form in waterand adopt a α-helical structure in organic solvents.14,31,37 Assuch, secondary structure appears as the single constant in theentry of this unique anionic CPP and amphipathic cationicCPPs. p28, like its amphipathic cationic counterparts, entersfrom a high dielectric medium (water) to a low dielectricmedium (core of the membrane). This suggests that thehydrophilic, cationic c-terminal 12 (p12) and hydrophobic n-terminal 16−18 amino acids (Figure 3B,C) distributed alongthe 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 cellmembrane and, second, assist in crossing the core of the cellmembrane through hydrophobic interaction.53 The initialincrease in α-helix and maintenance of the β-sheet content(Table 1) observed as solvent polarity is reduced, andsignificant increase in α-helicity and loss of β-sheet as it isreduced further also suggest that secondary structure may becritical for the translocation, if not the endocytosis of anionicCPPs.2 Although not quite as extensive in overall andpreferential entry into cancer cells, a similar behavior isobserved for amino acids 50 to 67 of p28 (p18) the initialhydrophobic α-helix and β-sheet of p28 and minimal motif(protein transduction domain) responsible for the preferentialentry of Azurin into human cancer cells.2 Although the netcharge of p28, p18, and p12 are all negative (−4, −2, and −2,respectively), the overall hydrophobic scores54 of p28, p18, andp12 are −0.35, 0.34, and −1.3, respectively. This suggests that areduction in hydrophobicity is reflected in the lack ofpreferential entry of p12.2 The reduction in overall andpreferential entry of p18, relative to p28, may also result fromthe lack of a hydrophilic, cationic α-helix reducing its entrypathway to that through endocytosis.2

Irrespective of route of entry, CD and Raman spectroscopyappear to be useful tools for predicting the entry ofamphipathic, anionic peptides. There was a uniform increasein the α-helical content of all L-p28 in TFE with bothapproaches, suggesting that as p28 approaches the cellmembrane its structure changes from largely random coil toα-helix while maintaining a relative stable degree of β-sheet,which decreases to essentially zero as an α-helical secondarystructure penetrates the bilipid membrane and traverses thecytoplasm, late endosomes, and ER to the nucleus.2 Thesignificant decrease and increase in the α-helix/β-sheet ratio ofp28 and D-substituted analogues, observed with CD and Ramanspectroscopy, respectively, provides a certain degree ofpredictability to the overall entry of a D-amino acid substitutedanionic CPP.Figure 2A,B shows that endocytotic penetration into cancer

cell lines is significantly higher than matched normal cells withsignificant entry (40−60%) occurring in both via a non-endocytotic pathway. This suggests that the degree ofendocytosis affects not only overall penetration but also the



preferential penetration of p28 into cancer cells (Figure 1). p28is an amphipathic α-helical peptide with a stretch ofhydrophobic amino acids predicted to be clustered about then-terminal α-helix and β-sheet.14 D-substitution of the n-terminal leucine of p28 does not alter the α-helix/β-sheet ratio(Table 1 and Figure 4D), overall entry, or intracellulardistribution (Figure 5), confirming that altering the chiralityof the initial amino acid in the n-terminal α-helix does not altereither endocytotic or direct entry into cells. The singlesubstitutions within the c-terminal at positions 22, 24, and 28of the p28 α-helix reduced the overall entry of these analoguesinto all cell types irrespective of the degree of overall entry. Insharp contrast, preferential penetration of all D-substitutedanalogues of p28 into cancer cells (fold increase over matchednormal cells) was essentially identical to that of p28 (Figure6B). This suggests that discrete changes in chirality can alteroverall entry, but not the preferential penetration ofamphipathic, anionic CPPs.In summary, although details for the mechanism that p28

and other anionic CPPs2 use to translocate a cell membraneremain unknown, it appears that the ratio of α-helix/β-sheetmotifs evolves as an anionic CPP transitions through the cellmembrane facilitating its translocation. The position of D-aminoacid substitutions within the α-helical backbone and thestrength of the substitutions that alter chirality38 may also becritical to the overall entry of a CPP, but not the preferentialnature of its penetration.

■ AUTHOR INFORMATION

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

NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTS

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

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