Article pubs.acs.org/Biomac
Apoptosis Inducing, Conformationally Constrained, Dimeric Peptide Analogs of KLA with Submicromolar Cell Penetrating Abilities Soonsil Hyun,†,§ Seonju Lee,‡,§ Seoyeon Kim,† Sangmok Jang,‡ Jaehoon Yu,*,† and Yan Lee*,‡ †
Department of Chemistry and Education and ‡Department of Chemistry, Seoul National University, Seoul 151-742, Korea S Supporting Information *
ABSTRACT: The apoptosis inducing KLA peptide, (KLAKLAK)2, possesses an ability to disrupt mitochondrial membranes. However, this peptide has a poor eukaryotic cell penetrating potential and, as a result, it requires the assistance of other cell penetrating peptides for effective translocation in micromolar concentrations. In an effort to improve the cell penetrating potential of KLA, we have created a library in which pairs of residues on its hydrophobic face are replaced by Cys. The double Cys mutants were then transformed to bundle dimers by oxidatively generating two intermolecular disulfide bonds. We envisioned that once transported into cells, the disulfide bonds would undergo reductive cleavage to generate the monomeric peptides. The results of these studies showed that one of the mutant peptides, dimer B, has a high cell penetrating ability that corresponds to 100% of fluorescence positive cells at 250 nM. Even though dimer B induces disruption of the mitochondrial potential and cytochrome c release followed by caspase activation at submicromolar concentrations, it displays an LD50 of 1.6 μM under serum conditions using HeLa cells. Taken together, the results demonstrate that the strategy involving formation of bundle dimeric peptides is viable for the design of apoptosis inducing KLA peptide that translocate into cells at submicromolar concentrations.
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INTRODUCTION Resistance to apoptosis is a hallmark of cancer.1 Because they are the central regulator of apoptotic cell death, mitochondria are universal targets in the development of drugs to treat all types of cancers. Pro-apoptotic small molecules, such as paclitaxel,2 doxorubicin,3 and ceramide,4 are known to exhibit mitochondria targeted cytotoxicity.5 Unlike small molecules, cationic amphipathic peptides bind to mitochondria and, by doing so, depolarize the mitochondrial membrane potential of cancer cells.6−8 Furthermore, owing to differences in their potentials, mitochondrial membranes are disrupted by charged peptides at lower concentrations than those required to disrupt plasma membranes.9 Because the lipid composition of mitochondrial membranes closely resemble those in Gramnegative bacteria,10 a variety of peptides that have antimicrobial activity against Gram-negative bacteria could also be effective in causing depolarization and/or disruption of mitochondrial membranes and acting as apoptosis-inducing agents. However, owing to their poor cell penetrating potentials, the charged peptides would be effective only at high micromolar concentrations, a level at which unfortunately they have the potential of interacting with other intracellular targets. The most actively investigated member of the family mitochondria disrupting peptides,11 KLA peptide (KLAKLAKKLAKLAK, Figure 1), has an α-helical structure when located in the membrane.12 From the time of the discovery of the proapoptotic ability of KLA, many efforts have been carried out to improve the efficacy of this protein by improving its cell © XXXX American Chemical Society
penetrating ability. The most straightforward approach taken to solve this problem involves the construction of hybrid peptides through linear conjugation of KLA with well-known cell penetrating peptides (CPPs) such as R7,13 penetratin,14 and MIIYRDLISH.15 Hybrid peptides formed by conjugation of KLA with cancer homing peptides16 and small molecules17 have also been described.18 In addition, replacements of hydrophilic face Lys by Arg residues19 and of hydrophobic face Leu residues by sterically more bulky hydrophobic analogs20 have been carried out in order to increase the eukaryotic cellular uptake of KLA. Although these investigations have led to significant improvements in efficacy, micromolar concentrations of the optimized KLA related peptides are still required to bring about apoptotic cell death. The stapling strategy has been used in the past to increase αhelical propensities of peptides as well as to produce conformationally constrained and chemically more stable analogs.21 The strategy has also been applied to solve cellular uptake problems because it is known that intracellular targeting amphipathic peptides with high α-helical contents have enhanced cell penetration activities.22 However, owing to the fact that they are constructed by covalently linking the component peptides, stapled peptides often do not retain the important biological properties of the original peptide. Received: July 17, 2014 Revised: August 25, 2014
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dx.doi.org/10.1021/bm501026e | Biomacromolecules XXXX, XXX, XXX−XXX
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Figure 1. Schematic representation of the strategy for intracellular delivery of apoptosis inducing KLA peptide using dimeric analogs (left). Helical wheel representations of KLA and the dimers are shown (right). Lowercase letters indicate positions in the heptad repeat associated with coiled−coil protein sequences (abcdefg) with hydrophobic residues at the a and d sites, which formed the hydrophobic core of the coiled−coil helical bundle. The a or d positions targeted for mutagenesis are highlighted in bold and are underlined. Polar Lys residues, nonpolar Leu and Ala residues, and Cys residues are shown in black, white, and gray circles, respectively. Disulfide bonds are indicated by the gray lines. were purchased from WelGENE (U.S.A.). Trysin-EDTA and OptiMEM were purchased from Life Technologies (U.S.A.). Peptide Synthesis. Solid Phase Peptide Synthesis. Peptide syntheses were performed by standard Fmoc solid phase synthesis using a manual microwave peptide synthesizer (CEM, U.S.A.). All synthesis steps were followed the same as previously26 except for Fmoc deprotection and amino acid coupling. For Fmoc deprotection, the resins were placed in a microwave vessel and irradiated for 2 min (ramping time for 1 min) at 5 W power. For the coupling step, the resins were microwave irradiated for 5 min (ramping time for 2 min) at 5 W power. The temperature was set at 35 °C for both steps. Dimerization of Peptides. Dimeric bundle peptides were prepared by air oxidation as previously described.24 Briefly, cysteinyl peptide monomer was dissolved in 0.1 M deaerated ammonium bicarbonate to give a final concentration of ∼1 mg/mL and the mixture was incubated to stand open to atmosphere until the reaction was complete. Parallel and antiparallel dimers were obtained and shown to be separated by HPLC using a C18 column (Zorbax C18, 3.5 mm, 4.6 × 150 mm) as the stationary phase and buffer A (water with 0.1%, v/v TFA) and buffer B (acetonitrile with 0.1%, v/v TFA) as the mobile phase. The gradient conditions of the mobile phase were as follows: 5 min, 5% B followed by linear gradient 5−70% B over 25 min. Parallel dimers are major products (antiparallel dimers were obtained less than 5% judged by HPLC traces) and found to be relatively nonpolar than antiparallel minor dimers as described previously.24 Dimeric peptides were confirmed by using MALDI-TOF and purified by a preparative HPLC. Fluorescence Labeled Peptides. The dye 5-TAMRA (Merck Millipore) was used to lead fluorescently labeled peptides. This fluorescent dye was amide coupled with peptides at N-terminus using 2-(6-choloro-1-H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminum hexafluorophosphate (HCTU) activation. Briefly, 5-TAMRA (2 eq, relative amount to Fmoc deprotected N-terminus amine) was dissolved in anhydrous dimethylformamide (DMF) to a final concentration of 0.1−0.5 M and activated with HCTU (2 equiv), 1-hydroxybenzotriazole (HOBt, 2 equiv), and diisopropylethylamine (4 equiv). The activated 5-TAMRA solution was added to the Fmoc deprotected resin and stirred for 2 h at room temperature. When the reaction was complete, peptides were cleaved from resins followed by the normal procedure. For dimer peptides, 5-TAMRA was labeled on only one strand. All peptides were confirmed by using a Voyager MALDI-TOF mass spectrometer (Applied Biosystems). KLA MS [M + H]+: 1565.1 (calcd), 1564.0 (obsd). Monomer A MS [M + H]+: 1544.9 (calcd), 1544.1 (obsd). Monomer B MS [M + H]+: 1629.0 (calcd), 1628.1 (obsd). Monomer C MS [M + H]+: 1587.0 (calcd), 1585.7 (obsd). Dimer A MS [M + H]+: 3084.9 (calcd), 3084.5 (obsd). Dimer B MS
Consequently, a useful strategy in designing stapled peptides that express the original function of the key peptides would be to utilize a linking technique that only temporarily bonds the component peptides. Thus, if the bond used for temporary stapling were readily cleaved following transport of the linked peptides into cells, the strategy would be applicable to intracellular delivery of peptides that are active against intracellular targets. In earlier studies, we utilized disulfide bonds, which are frequently used to produce conformationally constrained proteins and peptides23 in the design of dimeric helical peptides that bind to intracellular RNA targets.24 The dimers produced in the earlier effort consist of two α-helical peptides linked by two disulfide bonds, which temporarily constrain the structure. We observed that in contrast to the corresponding monomers the helical peptide dimers have dramatically improved cell penetration activities as exemplified by their highly efficient cellular uptake in eukaryotic cells even at low nanomolar concentrations.25 Moreover, once the dimeric proteins are transported through the plasma membrane, their disulfide bonds undergo ready, glutathione-promoted cleavage to generate the corresponding monomers, which serve as low nanomolar inhibitors of the Tat-TAR interaction of HIV-1. In the investigation described below, we have employed the novel disulfide bond-based strategy in the design of temporarily linked bundle dimeric peptides that consist of two close analogs of the apoptosis-inducing peptide KLA. The results of this study show that one of the dimeric peptides has a superior cell penetration ability and that once inside the cell it undergoes S− S bond cleavage to form the individual KLA analogs. Moreover, we have demonstrated that the dimeric peptides serve as low concentration acting, chemotherapeutic agents that induce apoptosis by depolarizing the mitochondrial membrane potential after internalization within cells (Figure 1).
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EXPERIMENTAL SECTIONS
Cell Line and Reagents. HeLa cells were cultured under standard condition in Dulbecco’s modified Eagle’s medium (DMEM, HyClone, Thermo Scientific) supplemented with 10% fetal bovine serum (FBS, HyClone, Thermo Scientific) and antibiotics (100 μg of streptomycin/ mL and 100 IU of penicillin/mL, Life Technologies). DPBS and FBS B
dx.doi.org/10.1021/bm501026e | Biomacromolecules XXXX, XXX, XXX−XXX
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[M + H]+: 3253.0 (calcd), 3253.4 (obsd). Dimer C MS [M + H]+: 3168.9 (calcd), 3169.4 (obsd). 5-TAMRA-KLA MS [M + H]+: 1936.2 (calcd), 1935.2 (obsd). 5-TAMRA-monomer A MS [M + H]+: 1916.1 (calcd), 1915.8 (obsd). 5-TAMRA-monomer B MS [M + H]+: 2000.2 (calcd), 1999.2 (obsd). 5-TAMRA-monomer C MS [M + H]+: 1958.1 (calcd), 1957.4 (obsd). 5-TAMRA-dimer A MS [M + H]+: 3456.0 (calcd), 3457.9 (obsd). 5-TAMRA-dimer B MS [M + H]+: 3624.2 (calcd), 3625.2 (obsd). 5-TAMRA-dimer C MS [M + H]+: 3540.1 (calcd), 3541.7 (obsd). 5-TAMRA-R7 MS [M + H]+: 1521.8 (calcd), 1522.4 (obsd). 5-TAMRA-penetratin MS [M + H]+: 2657.4 (calcd), 2657.7 (obsd). The concentrations of peptide stock were measured by Direct Detect spectrometer (Millipore, U.S.A.). For 5-TAMRA labeled peptides, extinction coefficients were used (ε260 = 32 300, ε556 = 89 000 L mol−1 cm−1).27 Circular Dichroism Spectroscopy. α-Helicities of peptides were measured using a Chirascan plus Circular Dichroism detector (Applied Photophysics) with 0.05 cm path-length cell. CD spectra were scanned from 190 to 260 nm with 0.2 s integration, 1 nm step resolution, and 1 nm bandwidth. Three scans were performed and averaged. Peptides were dissolved in 10 mM sodium phosphate (pH 7.4) to a final concentration of 20 μM and scanned at 20 °C (Supporting Information Figure S2A,B) or 37 °C (Supporting Information Figure S2C,D). Because of the detection limit of the CD spectrometer, the αhelicities of peptides were measured at 20 μM, which is a higher concentration than those applied to cells. For α-helix inducing condition, 50% 2,2,2,-trifluoroethanol (TFE, Sigma-Aldrich) was used in the same buffer. To calculate α-helicities of peptides, the averaged CD spectra (ranging from 200 to 260 nm) were analyzed using CDNN secondary structure analysis software (version 2.1) authored by Gerald Böhm at the Institute for Biotechnology, Martin Luther University, Halle-Wittenberg (Germany). The α-helicities of peptides are summarized in Table 1.
Hydrodynamic Size Measurement. The peptide solutions in DPBS were incubated at ambient temperature for 30 min. Size distributions were measured by Zetasizer 3500 (Malvern Instruments, U.S.A.) equipped with a He−Ne ion laser at a wavelength of 633 nm. Cytochrome c Release. Isolation of Mitochondria. Mitochondria were isolated from HeLa cell using a dounce homogenizer following a modified previous protocol.28 Briefly, ca. 3.0 × 107 HeLa cells were harvested and washed once with ice cold phosphate-buffered saline (PBS). Cells were resuspended in a freshly prepared isolation buffer (225 mM mannitol, 75 mM sucrose, 0.04 mM EGTA, and 30 mM Tris−HCl pH 7.4, 2 mL). The suspension of HeLa cells were placed in a precooled dounce homogenizer and lysed by ca. 70 times of slow strokes. Lysis efficiency was monitored by visual estimation using a microscope. The homogenate was transferred to precooled microcentrifuge tubes on ice and centrifuged at 600g for 5 min at 4 °C. Supernatant was centrifuged at 7000g for 10 min at 4 °C and mitochondrial fraction was recovered. The isolated mitochondrial fraction was resuspended in a freshly made mitochondria buffer (225 mM mannitol, 75 mM sucrose and 30 mM Tris−HCl (pH 7.4)) and used for the assay. Cytochrome c Release in Isolated Mitochondria. The designated concentrations of peptides were incubated with mitochondria fraction (2 mg/mL) in a mitochondria swelling buffer (150 mM KCl, 5 mM sodium succinate, 0.5 mM KH2PO4, 5 mM Tris-HCl (pH 7.4), and 2.5 μM of rotenone) for 20 min at room temperature at the 25 μL scale and centrifuged at 13 000 rpm for 10 min at 4 °C. The supernatant was removed and the pellet was dissolved with phosphate buffered saline (PBS) containing 0.1% Tween 20. The supernatant and pellet fractions were subjected to fractionation on a 12% SDS-PAGE and transferred to PVDF membrane (Millipore). After blocking for 30 min at room temperature with 5% bovine serum albumin (BSA) in PBS containing 0.1% Tween 20, blots were incubated overnight at 4 °C with mouse monoclonal anticytochrome c mouse (K257-100-5, BD Bioscience, 1:500 diluted in PBS containing 0.1% Tween 20 and 5% BSA,) as primary antibody and horseradish peroxidase-linked goat antimouse secondary antibody (sc-2055, Santa Cruz Biotechnology, 1:2000 in PBS containing 0.1% Tween 20 and 1% BSA) for 1 h at room temperature. The immunoreactive proteins were detected by Luminata crescendo western HRP substrate (Millipore), using X-ray film exposure. Cytochrome c Release in Intact Cells. HeLa cells (5.0 × 104 cells) were incubated in the presence and absence of peptides and harvested. To separate the cytosolic fraction from other cellular components, cells were lysed for 10 min on ice in a buffer containing 20 mM HEPES, pH 7.2, 50 mM KCl, 5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 250 mM sucrose, 200 μg/mL digitonin, and protease inhibitor (Sigma-Aldrich). Cells were centrifuged at 13 000 rpm for 5 min at 4 °C and the supernatant was removed as the cytosolic fraction. The pellet as the mitochondrial fraction was solubilized in radioimmune precipitation assay (RIPA) buffer (150 mM NaCl, 0.1% sodium dodecyl sulfate, 0.5% sodium deoxycholate, 1% NP-40, 50 mM TrisHCl, pH 8.0, and protease inhibitor). Each cytosolic and mitochondrial fraction were used for Western blot. β-Actin or Cox IV served as internal loading control to normalize the expression of proteins. The following antibodies were used: mouse monoclonal antiβ-actin (Catalog: 3700, Cell Signaling Technology, 1:2,000), rabbit monoclonal anti-Cox IV (Catalog: 5247, clone 3E11, HRP conjugate, Cell Signaling), and goat antimouse IgG HRP-conjugated secondary antibody (SC-2055; Santa-Cruz Biotechnology, Santa Cruz, CA) JC-1 Mitochondrial Membrane Potential Assay. Mitochondria Staining Kit (Catalog: 89874, Sigma-Aldrich) was used to detect changes of mitochondrial potential and cells were stained followed by the manufacturer’s protocol. Confocal Microscopy. HeLa cells (1.0 × 104 cells/well) seeded on 8-well Lab-Tek chamber slides (Thermo) were stained with JC-1 dye. Green and red fluorescence (488 and 555 nm emission wavelength) of JC-1 stained cells were visualized using a Confocal LSM 710 system (Zeiss) equipped with argon and helium−neon lasers (Carl Zeiss). Images were captured using Axiocam camera equipped with the
Table 1. Sequences and Helicities of KLA and Dimers A-C
α-Helicities of the dimers were measured in 10 mM sodium phosphate (first value) and in 50% TFE in the same buffer (second value) at 20 °C. a
Cell Penetration Efficiency. HeLa cells (1.5 × 105 cells/well) were seeded in 24-well plate in DMEM containing 10% FBS. After 24 h incubation, 50 μL of peptide dispersions with various concentrations in Opti-MEM were prepared and added into the cells for 4 h. For the assay, the cells were washed with DPBS and trypsinized. Detached cells were collected and centrifuged at 13 000 rpm for 10 min and resuspended in DPBS containing 1% FBS. The FACS analysis was performed on FACSCalibur (Becton Dickinson, U.S.A.) with the excitation wavelength of 488 and 585 nm emission filter. A total of 20 000 cells were assessed for each sample and dead cells were excluded from the analysis. For cellular uptake mechanism study, cells were pretreated with wortmannin (50 μg/mL), amiloride (15 μg/mL), and nystatin (50 μg/ mL, all from Sigma-Aldrich) for 2 h and 50 μL of peptide dispersion (final concentration of 250 nM) were added into cells for 4 h. C
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appropriate wavelength of filters and using identical settings and exposure times. Flow Cytometry. HeLa cells (5.0 × 104 cells/well) were seeded on a 24 well plate and incubated for 12 h. Cells were incubated further in the presence of peptides for 4 h, then harvested and dissolved in 0.3 mL PBS to measure the green fluorescence (FITC filter) and red fluorescence (PI filter) by flow cytometry (BD FACSCanto II, BD Bioscicence) and mean fluorescence intensity was measured and analyzed. Caspase-3 Activity Assay. HeLa cells (2.0 × 105 cells/well) were seeded in 24 well plate in DMEM containing 10% FBS. After 24 h incubation, peptides were added and cells were incubated for 24 h. The cells were washed with DPBS and trypsinized. Detached cells were collected and counted. Then collected cells were centrifuged at 13 000 rpm for 10 min, and the pellets were lysed in cell lysis buffer on ice for 30 min. The lysed cells were recentrifuged at 13 000 rpm for 10 min and the lysates were analyzed by using a colorimetric caspase-3 assay kit (Catalog CASP3C, Sigma-Aldrich). The lysates were incubated with caspase-3 substrate in humidified incubator at 37 °C for 2 h. The absorbance was measured at 405 nm using a microplate reader (Molecular Devies, CA). Cell Viability Assay. HeLa cells were seeded on a 96-well plate at a density of 5.0 × 103 cells/well. After 24 h, various concentrations of peptides were treated in a fresh complete media containing 10% FBS for 24 h. Cell viability was measured using a WST-1 reagent (EZCytox, Daeillab, Korea).
Figure 2. Cell penetration efficiency of 5-TAMRA-dimers A−C measured by using FACS. (a) Cell penetration efficiency was calculated as a percentage of fluorescence-positive cells at concentrations of 250 nM. (b) Concentration dependence of cellular uptake of 5-TAMRA-dimer B. On the other hand, 5-TAMRA-dimer A showed 39 ± 9% of cell uptake at 2 μM. Each data point represents the average value of three experiments (±SD).
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RESULTS AND DISCUSSION Synthesis of Dimeric Bundle Peptides and their αHelicities. Employing the disulfide bond based strategy described above, we designed three dimeric bundle peptides that are generated by intermolecular S−S bond forming reactions of three KLA Cys mutants. In the KLA mutants, hydrophobic amino acids Leu at the 2- and 9-positions and Ala at the 6- and 13-position are replaced by Cys residues. The three mutant peptides, monomers A−C, each containing an Nacetyl capping group, are generated using standard solid phase peptide synthesis via Fmoc chemistry. Air oxidation of the individual monomers then generates the respective dimers A− C, which contain S−S bonds between their 2,2′ and 9,9′ (dimer A), 6,6′ and 13,13′ (dimer B), and 2,2′ and 13,13′ (dimer C) positions (Table 1, wheel diagram in Figure 1). Air oxidation of these peptides produces parallel dimers as major products.24 Fluorescence labeled counterparts of these bundle dimers were also synthesized by using 5-TAMRA N-capped analogs of monomer A−C. Following HPLC purification, dimers A−C both in aqueous solution and under membrane conditions were subjected to circular dichroism (CD) measurements in order to determine their α-helical contents. The results (Table 1, Supporting Information Figure S2) demonstrate that unlike previously studied LK dimers, which have similar α-helical contents irrespective of the positions of the disulfide bonds,24 dimers A−C have different α-helical propensities with dimers B and C having high and near equal α-helicities and dimer A having the lowest α-helical content. The α-helical contents of dimer B are almost maintained even at the physiological temperature, 37 °C. Cell Penetration of Dimeric Bundle Peptides. The HeLa cell penetrating potentials of the bundle dimers were measured by using FACS with 5-TAMRA-labeled dimers A−C and monomers A−C (Figure 2). 5-TAMRA-R7 and 5TAMRA-penetratin were employed as reference cell penetration peptides. The results of these measurements show that cellular uptakes of dimers B and C at 250 nM concentrations are much greater than those of dimer A, KLA and the reference
peptides at the same concentrations (Figure 2a). The findings confirm the prediction that transport through the plasma membrane is enhanced by incorporating the KLA peptide platform into a bundle dimeric form. The cell penetrating ability of dimer B was evaluated at varying concentrations (Figure 2b). Remarkably, dimer B displays a marked increase in its cell penetration efficiency starting at ca. 50 nM and it penetrates into over 90% of cells at 125 nM. The monomeric peptides, dimer A, and the other cell penetration peptides display high cell penetration efficiencies only when used in micromolar concentrations. The combined findings demonstrate the validity of the prediction that transport through the plasma membrane is enhanced by incorporating the KLA peptide platform into a bundle dimeric form. Although the enhanced cell penetration of dimers B and C can be rationalized in part by their higher α-helical contents, another reason needs to be found to explain the higher cell penetration potential of dimer B over dimer C at low concentrations. Additional studies showed that at 250 nM dimer B, but not dimers A and C and monomers A−C, forms aggregates that have sizes up to several hundred nanometers (Figure 3a). It is possible that the relatively higher hydrophobicity of dimer B, which unlike the others contains four Leu residues, might be the reason why it exclusively forms the aggregates. An investigation of the concentration dependence of the hydrodynamic sizes of the dimer B derived aggregates revealed that a dramatic increase in size occurs at ca. 50 nM (Figure 3b), the same concentration at which the cell penetration efficiency of dimer B increases abruptly. This observation suggests that dimer B possesses a lower critical aggregation concentration than the other dimers and that the generated nanosized aggregates are the species internalized into cells. Thus, the overall findings suggest that high α-helicity of dimeric peptides is the essential factor for efficient cell D
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cytochrome c release from mitochondria.28 Because dimer B is readily reduced to form the corresponding monomer B under cytosolic conditions, we expected that cytochrome c release is actually promoted by monomer B. This expectation was confirmed using isolated mitochondria (Figure 5a). Original
Figure 3. Hydrodynamic size of KLA and dimers A−C measured by using DLS. (a) Comparison of hydrodynamic size of KLA and dimers A−C at concentrations of 250 nM. (b) Hydrodynamic size change of dimer B according to concentration change. All measurements were performed in DPBS (pH 7.4). Each data point represents the average value of three experiments (±SD).
Figure 5. Mitochondrial membrane disruption study. Released cytochrome c was detected by Western blot. (a) Mitochondria fraction isolated from HeLa cells were incubated with peptides or Ca2+ for 20 min at room temperature. Mitochondria were centrifuged and the cytochrome c was detected from supernatant as released and from the mitochondrial pellet as retained. (b) Dimer B induced release of cytochrome c in intact cells. HeLa cells were treated with designated concentrations of KLA or dimer B for 4 h. Cytosolic fraction and the pellet containing mitochondria were subjected to SDS-PAGE and immunoblotted with cytochrome c and β-actin antibodies as loading control for cytosol (or COX IV antibody as loading control for pellet fractions). Data are representative of at least three experiments.
penetration and the aggregation can boost the cell penetration more efficiently.29−31 The mechanism for cell penetration by dimer B was explored using various endocytosis inhibiting conditions (Figure 4). At
KLA peptide, dimer B, and monomer B induced the release of cytochrome c at 1 μM. Even though cytochrome c release reaches a maximum value in the presence of 1 μM of monomer B, it occurs even when the monomer concentration is less than 1 μM. Interestingly, when intact HeLa cells were treated with peptides, dimer B could induce the release of cytochrome c at 0.1 μM and maximize the release at 1 μM, whereas KLA peptide showed only limited release even at 10 μM (Figure 5b). The finding suggests the mitochondrial membrane is disrupted by monomer B produced from dimer B that is translocated into cells at submicromolar concentrations. Loss of mitochondrial membrane potential caused by dimer B was probed using confocal microscopy with JC-1 stained HeLa cells (Figure 6a).32 Red fluorescence of JC-1 changed into green by depolarization of mitochondrial membrane. Specifically, significant depolarization was observed in the images of dimer B treated cells. At more than 5 μM, significant morphological changes were also observed. Membrane depolarization promoted by various peptides was quantitated by measuring red/green ratios, determined using flow cytometry (Figure 6b). The data show that depolarization occurs at less than 1 μM of dimer B but that KLA and monomer B do not depolarize mitochondria even at concentrations as high as 10 μM. The result also strongly supports the strong cell penetrating capability of dimer B over other reference peptides.
Figure 4. Mechanism for cellular uptake of 5-TAMRA-dimer B at 250 nM. Each data point represents the average value of three experiments (±SD).
low temperature, cell penetration by this dimer is almost completely inhibited, indicating that an energy dependent transport pathway is followed. In addition, cell penetration of dimer B was not blocked by amiloride, an inhibitor of macropinocytosis, whereas it is significantly disrupted by wortmannin, an inhibitor of macropinocytosis and clathrinmediated endocytosis, and by nystatin, an inhibitor of caveolaemediated endocytosis. The results demonstrate that dimer B is internalized in cells mainly through energy-dependent endocytic pathways mediated by clathrin or caveolae. Induction of Apoptosis by Dimeric Bundle Peptides. In the key phase of this effort, we observed that treatment of cells with submicromolar concentrations of dimer B initiates early apoptotic signals caused by disruption of the mitochondrial membrane. Mitochondrial membrane disruption by the peptides was quantified by using Western blot analysis of E
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Figure 8. Relative cell viability of HeLa cells treated with KLA peptides. HeLa cells (5000/well) were incubated with peptides for 24 h at concentration ranging from 0 to 100 μM. Cell viability was determined with WST-1 assay. Mean values (±SD) were calculated from at least three independent experiments.
However, a discrepancy exists between the effective concentration of cellular uptake (EC50 = 50 nM) and the effective concentration of apoptotic cell death (LD50 = 1.6 μM) of dimer B. Thus, it appears that a threshold concentration of cytochrome c, which is released upon disruption of the mitochondrial membrane by the peptide, is required in order to bring about cell death. This observation parallels those made in several previous studies,33−35 which suggest that micromolar concentrations of cytochrome c in the cell are needed for the promotion of apoptosis induced cell death.
Figure 6. (a) Confocal microscopy images of JC-1 stained HeLa cells. Cells incubated for 4 h with peptides were stained with JC-1 dye. (b) Quantitative analysis of the red/green ratio in untreated and peptide treated cells measured by flow cytometry. The values are expressed as a ratio of that obtained in untreated cells. Data are shown as the mean values ± SD of triplicates. Not significant (N.S.) P > 0.05, *P < 0.01, **P < 0.001 (Student’s t test).
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CONCLUSION In the effort described above, we have shown that KLA peptide, which has the potential to disrupt mitochondrial membrane but lacks the ability to translocate efficiently into cells, can be transformed into dimeric bundle peptides by replacement of two hydrophobic residues by two Cys followed by oxidative S− S bond formation. Among the three bundle dimers created using this strategy, dimer B, which possesses disulfide linkages between the 6,6′ and 13,13′ positions of the component KLA bis-Cys mutants, was found to have the highest HeLa cell penetrating ability. Highly efficient uptake of this dimer into cells takes place at a concentration of 250 nM. Following translocation into cells, dimer B is reductively converted into monomer B, which then promotes disruption of the mitochondrial membrane. Dimer B showed more than 2 orders of magnitude improved apoptotic potential relative to KLA peptide. The significance of the results of this investigation are heightened by the fact that they demonstrate the generality of the temporarily constrained peptide strategy in applications to the construction of efficient cell penetrating dimeric peptides that release active monomeric peptides once inside cells.
In order to determine if the dimeric peptides promote a late apoptosis signal, the abilities of dimers A and B to stimulate caspase-3 activity were assessed (Figure 7). The results
Figure 7. Comparison of caspase-3 activities of dimer A and B at 250 nM. (N.S. P > 0.05, *P < 0.01 (Student’s t test)). Each data point represents the average value of three experiments (±SD).
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demonstrate that cells treated with 10 μM dimer B display a 2-fold higher caspase-3 activity as compared to that of untreated cells whereas those treated with 10 μM dimer A have an activity that is similar to untreated cells. Apoptosis was clearly induced by dimer B with high cell penetrating potential. Finally, the cytotoxicities of the dimers and KLA peptide were determined by using WST-1 assay. The LD50 of dimer B was found to be 1.6 μM (Figure 8), a value that is about 2 orders of magnitude lower than that of KLA peptide, which displays higher than 100 μM under the same conditions.
ASSOCIATED CONTENT
S Supporting Information *
Chromatograms and CD spectra of synthesized peptides and confocal microscopy images of control cells. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Authors
*E-mail: (J.Y.)
[email protected]. *E-mail: (Y.L.)
[email protected]. F
dx.doi.org/10.1021/bm501026e | Biomacromolecules XXXX, XXX, XXX−XXX
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Author Contributions
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These authors contributed equally.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was financially supported by a grant (20120008760) from National Research Foundation of Korea, and the GAIA project (G113-00055-3004-0) funded by Ministry of Environment, Korea, and a project (KDDF-20140407) from Korea Drug Development Fund.
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ABBREVIATIONS DLS, dynamic light scattering; DPBS, Dulbecco’s phosphate buffered saline; FACS, fluorescence activated cell sorting; Fmoc, fluorenylmethyloxycarbonyl; TAMRA, carboxytetramethylrhodamine; WST-1, water-soluble tetrazolium-1
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dx.doi.org/10.1021/bm501026e | Biomacromolecules XXXX, XXX, XXX−XXX
