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.
■
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
© 2014 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
Published: September 4, 2014
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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).
■
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
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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.
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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).
■
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
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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
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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).
■
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).
■
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.
■
AUTHOR INFORMATION
Corresponding Authors
*E-mail: (J.Y.) jhoonyu@snu.ac.kr.
*E-mail: (Y.L.) gacn@snu.ac.kr.
3751
dx.doi.org/10.1021/bm501026e | Biomacromolecules 2014, 15, 3746−3752
Biomacromolecules
Article
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§
These authors contributed equally.
Notes
The authors declare no competing financial interest.
■
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.
■
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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