Cell-permeable, mitochondria-targeting, antioxidant peptides Cell-Permeable Peptide Antioxidants ...

37 Pages·2004·0.58 MB·English

JBC Papers in Press. Published on June 2, 2004 as Manuscript M402999200
Cell-permeable, mitochondria-targeting, antioxidant peptides

Cell-Permeable Peptide Antioxidants targeted to Inner Mitochondrial Membrane inhibit
Mitochondrial Swelling, Oxidative Cell Death and Reperfusion Injury

Kesheng Zhao1, Guo-Min Zhao1, Dunli Wu1, Yi Soong1, Alex V. Birk2, Peter W. Schiller3 and
Hazel H. Szeto1
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1Department of Pharmacology and 2Department of Biochemistry, ed
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Joan and Sanford I. Weill Medical College of Cornell University http
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1300 York Avenue, New York, N.Y. 10021 w
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3Laboratory of Chemical Biology and Peptide Research brg/
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Clinical Research Institute of Montreal es
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110 Pine Avenue West, Montreal, Quebec, Canada H2W 1R7 nu
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Corresponding author: Hazel H. Szeto, M.D., Ph.D.
Department of Pharmacology
Weill Medical College of Cornell University
1300 York Avenue, New York, N.Y. 10021
Tel: [phone]; Fax: [phone]
Email: [email protected]
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Copyright 2004 by The American Society for Biochemistry and Molecular Biology, Inc.
Cell-permeable, mitochondria-targeting, antioxidant peptides
SUMMARY
Reactive oxygen species (ROS) play a key role in promoting mitochondrial cytochrome c
release and induction of apoptosis. ROS induce dissociation of cytochrome c from cardiolipin
on the inner mitochondrial membrane (IMM), and cytochrome c may then be released via
mitochondrial permeability transition (MPT)-dependent or MPT-independent mechanisms. We
have developed peptide antioxidants that target the IMM, and used them to investigate the role of
ROS and MPT in cell death caused by t-butylhydroperoxide (tBHP) and 3-nitropropionic acid
(3NP). The structural motif of these peptides centers on alternating aromatic and basic amino
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acid residues, with dimethyltyrosine (Dmt) providing scavenging properties. These peptide ow
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antioxidants are cell-permeable and concentrate 1000-fold in the IMM. They potently reduced ed
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intracellular ROS and cell death caused by tBHP in neuronal N2A cells (EC50 ~ nM). They also http
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decreased mitochondrial ROS production, inhibited MPT and swelling, and prevented w
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cytochrome c release induced by Ca2+ in isolated mitochondria. They also inhibited 3NP- brg/
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induced MPT in isolated mitochondria, and prevented mitochondrial depolarization in cells es
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treated with 3NP. ROS and MPT have been implicated in myocardial stunning associated with nu
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reperfusion in ischemic hearts, and these peptide antioxidants potently improved contractile force , 2
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in an ex vivo heart model. Importantly, peptide analogs without Dmt did not inhibit
mitochondrial ROS generation or swelling, and failed to prevent myocardial stunning. These
results clearly demonstrate that overproduction of ROS underlies the cellular toxicity of tBHP
and 3NP, and ROS mediate cytochrome c release via MPT. These IMM-targeted antioxidants
may be very beneficial in the treatment of aging and diseases associated with oxidative stress.
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Cell-permeable, mitochondria-targeting, antioxidant peptides
The mitochondrial respiratory chain on the inner mitochondrial membrane (IMM) is a major
intracellular source of reactive oxygen species (ROS). ROS cause non-specific damage to lipids,
proteins and DNA, leading to alteration or loss of cellular function. Mitochondria are
continuously exposed to ROS and accumulate oxidative damage more rapidly than the rest of the
cell, especially since ROS are highly reactive and short-lived (1). Many studies have associated
mitochondrial dysfunction caused by ROS to both necrotic and apoptotic cell death (2). The rate
of mitochondrial ROS production can be altered by several physiological or pathological
conditions. Inhibitors of the respiratory chain such as 3-nitropropionic acid (3NP), an
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irreversible inhibitor of the complex II enzyme succinate dehydrogenase, tend to increase ROS ow
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production (3-5). The inhibition of this complex appears to be related to neuronal death similar ed
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to those occurring in Huntington’s disease (6), and antioxidants can attenuate the neurochemical http
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changes and some behavioral disturbances caused by 3NP in animals (5;7). Mitochondrial Ca2+ w
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is another powerful signal for ROS production. Calcium is taken up into mitochondria via a brg/
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uniporter in the IMM, and elevation of mitochondrial Ca2+ and ROS production is thought to es
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play an important part in cell death associated with ischemia-reperfusion as well as 3NP (4;5). nu
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Increasing evidence suggest that ROS play a key role in promoting cytochrome c release , 2
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from the mitochondria (8-11), and cytochrome c in the cytoplasm triggers activation of the
caspase cascade that ultimately leads to apoptosis (12;13). The mechanism underlying ROS-
mediated cytochrome c release from mitochondria is still not fully understood. Cytochrome c is
normally bound to the IMM by an association with cardiolipin (14). It is now thought that
cytochrome c release from mitochondria proceeds by a two-step process – dissociation of
cytochrome c from cardiolipin in the IMM, followed by release of cytochrome c through the
outer mitochondrial membrane (OMM) (15). Cardiolipin is rich in unsaturated fatty acids and
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Cell-permeable, mitochondria-targeting, antioxidant peptides
peroxidation of cardiolipin induces the dissociation of cytochrome c from mitochondria into the
cytosol (16). However, the mechanism by which cytochrome c is released through the OMM is
not clear. One mechanism may involve ROS-induced promotion of Ca2+-dependent
mitochondrial permeability transition (MPT), with swelling of the mitochondrial matrix and
rupture of the OMM (17;18). ROS may promote MPT by causing oxidation of thiol groups on
the adenine nucleotide translocator (ANT)(19-21). This mechanism appears likely in 3NP
toxicity and ischemia-reperfusion injury where increased intracellular Ca2+ and ROS are both
present (4;5;22;23). However, there is also evidence showing that cytochrome c can be released
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through the OMM in a MPT-independent manner (24-28). It was recently reported that ROS can ow
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induce cytochrome c release from mitochondria in the absence of Ca2+ and was insensitive to ed
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cyclosporin A (10), suggesting MPT-independent mechanisms. MPT-independent mechanisms http
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may involve the voltage-dependent anion channel (VDAC) on the OMM or an oligomeric form w
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of Bax (15;25;29). brg/
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Given the many ways by which cytochrome c may be released through the OMM, the es
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most efficient approach to inhibit ROS-induced cytochrome c release and cell death would be nu
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prevention of lipid peroxidation of the IMM. Unfortunately, none of the available antioxidants , 2
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specifically target mitochondria, let alone the IMM. In addition, most of the antioxidants are
poorly cell-permeable, requiring concentrations in excess of 100 µM to prevent oxidative cell
death. One approach used to target antioxidants such as coenzyme Q and vitamin E to
mitochondria has involved conjugation of these lipid-soluble molecules to lipophilic cations such
as triphenylalkylphosphonium ions (TPP+), which are rapidly taken up into the mitochondrial
matrix because of the potential gradient across the IMM (30;31). The introduction of cations
into the mitochondrial matrix, however, leads to dissipation of IMM potential, and this was
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observed in isolated mitochondria with concentrations of TPP+-conjugated antioxidants greater
than 20 µM (30;31). Furthermore, dissipation of the IMM potential would ultimately limit
further drug uptake.
We have developed a series of peptide antioxidants that are taken up by mitochondria and
concentrate in the IMM. These peptide antioxidants are cell-permeable and are very potent at
reducing intracellular ROS and preventing cell death caused by the oxidant t-butylhydroperoxide
(tBHP). We have used these IMM-targeted antioxidants to investigate the role of
mitochondrially-generated ROS in mitochondrial dysfunction in cells exposed to 3NP. To
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dysfunction, we used isolated mitochondria to determine their ability to prevent MPT and d
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cytochrome c release caused by Ca2+ overload and 3NP. In addition, since ROS have been http
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implicated in contractile dysfunction associated with reperfusion of ischemic hearts, we w
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determined the efficacy of these peptide antioxidants in preventing myocardial stunning in an ex brg/
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vivo perfused heart model. Finally, to prove that the effects of these peptide antioxidants are due st o
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to their ability to scavenge ROS, we designed a peptide analog that lacked antioxidant properties. u
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Our results suggest that overproduction of ROS underlies the cellular toxicity of tBHP and 3NP, , 2
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and ROS mediate cytochrome c release via MPT and rupture of the OMM. These results also
confirm a major role for ROS in mitochondrial dysfunction and reperfusion injury, and
demonstrate the therapeutic potential of these peptide antioxidants in ischemia-reperfusion injury
and neurodegeneration.

MATERIALS AND METHODS

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Chemicals and Reagents – The SS peptides are tetrapeptides with alternating aromatic
residues and basic amino acids. SS-02 (Dmt-D-Arg-Phe-Lys-NH ), SS-20 (Phe-D-Arg-Phe-Lys-
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NH ), SS-31 (D-Arg-Dmt-Lys-Phe-NH ), and [3H]SS-02 were synthesized as described
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previously (32;33). A fluorescent analog (SS-19, Dmt-d-Arg-Phe-atnDap-NH ) containing β-
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anthraniloyl-L-α,β-diaminopropionic acid in place of the Lys4 residue in SS-02 was prepared for
mitochondrial and cellular uptake studies (34). All other chemicals were obtained from Sigma-
Aldrich (St. Louis, MO).
Measurement of Antioxidant Properties of SS peptides in vitro – The ability of SS
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peptides to scavenge H O in vitro was determined using luminol chemiluminescence (35). w
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H2O2 (4.4 nmol) was incubated with 1 to 100 µM of various peptides in 0.5 ml phosphate buffer fro
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(pH 8.0) for 30 s. Luminol (25 µM) and horseradish peroxidase (HRP; 0.7 IU) were then added ttp
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to the solution and chemilumunescence was monitored with a Chronolog Model 560 .jb
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aggregometer (Havertown, PA) for 20 min at 37oC. Antioxidant properties of SS peptides were b/
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further established by inhibition of fatty acid peroxidation and low density lipoprotein (LDL) t o
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oxidation. Linoleic acid peroxidation was initiated with ABAP (2,2’-azobis(2-amidinopropane)) ary
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and the formation of conjugated dienes was monitored spectrophotometrically at 234 nm (36). 01
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Freshly prepared human LDL (0.1 mg/ml in PBS buffer) was oxidized catalytically by the
addition of 10 µM CuSO , and the formation of conjugated dienes was monitored at 234 nm for
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5 h at 37oC (37).
Mitochondrial Preparation - Male CD1 mice were sacrificed by decapitation, and the
livers immediately excised and homogenized in ice-cold isolation buffer (10 mM sucrose, 200
mM mannitol, 5 mM HEPES and 1 mM EGTA, pH 7.4) containing 1mg/ml fatty acid-free BSA.
The homogenate was centrifuged for 10 min at 900 x g and the supernatant centrifuged again at
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13,800 x g for 10 min. The mitochondrial pellet were washed twice, centrifuged at 11,200 x g
and re-suspended in the same buffer (no EGTA). All experiments were conducted in accordance
with guidelines approved by the Institution for the Care and Use of Animals at Weill Medical
College of Cornell University.
Mitochondrial Uptake Studies – Uptake of SS-19 by isolated mitochondria was examined
by fluorescence quenching upon addition of a mitochondrial suspension (0.35 mg) (Hitachi F-
4500 fluorescence spectrophotometer; ex/em = 320 nm/420 nm). For mitochondrial uptake of
[3H]SS-02, mitochondria (0.8 mg) were suspended in buffer (70 mM sucrose, 230 mM mannitol,
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3 mM Hepes, 5 mM succinate, 5 mM KH PO , 0.5 µM rotenone, pH 7.4) containing [3H]SS-02 ow
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and 1 µM SS-02 at room temperature. Uptake was stopped by centrifugation (16000 x g for 5 d fro
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min at 4oC), and the mitochondrial pellet resuspended in 0.2 ml 1% SDS/0.2 N NaOH, and ttp
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radioactivity determined. Mitochondrial uptake of SS-19 and [3H]SS-02 were also determined in w.jb
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the presence of 1.5 µM FCCP (carbonyl cyanide p-(trifluoromethoxy)-phenylhydrazone), an b/
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uncoupler that results in mitochondrial depolarization. To determine the localization of the t o
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peptide within mitochondria, three cycles of freeze-thaw treatment was used to isolate inner and a
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outer membranes (38). Treatment with 1% digitonin was used to disrupt the outer membrane in 0
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order to determine peptide distribution to the IMM and matrix (39).
Cell Culture - Caco-2 cells (American Type Culture Collection, Manassas, VA) and N A
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cells (provided by Dr. Gunnar Gouras, Department of Neurology, Weill Medical College of
Cornell University) were cultured as described previously (40;41). Cell culture supplies were
obtained from Invitrogen (Carlsbad, CA).
Cellular Uptake and Intracellular Localization of Peptide Antioxidants – Peptide uptake
into Caco-2 cells were carried out as described previously (40). Cells (106/well) were incubated
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with [3H]SS-02 at 37oC for 60 min, and radioactivity was determined in the medium and in cell
lysate. To determine intracellular peptide localization, Caco-2 cells were incubated with SS-19
(0.1 µM) for 15 min at 37oC, and confocal laser scanning microscopy (CLSM) carried out with
living cells using a C-Apochromat 63x/1.2W corr objective (Nikon, Tokyo, Japan) with
excitation/emission wavelengths set at 320/420 nm. To demonstrate localization of SS-19 to
mitochondria, Caco-2 cells were incubated with SS-19 and Mitotracker TMRM
(tetramethylrhodamine methyl ester)(Molecular Probes, Portland, OR; ex/em = 550/575 nm) for
30 min at 37oC and then examined by CLSM.
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Intracellular ROS and Cell Viability - N A cells were plated in 96-well plates at a density w
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of 1 x 104 /well and allowed to grow for 2 days before treatment with tBHP (0.5 or 1 mM) for 40 ed
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min. Cells were washed twice and replaced with medium alone or medium containing varying ttp
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concentrations of SS-02 or SS-31 for 4 h. Intracellular ROS was measured by carboxy- w
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H2DCFDA (Molecular Probes, Portland, OR). Cell death was assessed by a cell proliferation brg/
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assay (MTS assay, Promega, Madison, WI). st o
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Intracellular Mitochondrial Potential - Caco-2 cells were treated with 3NP (10 mM) in u
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the absence or presence of SS-02 (0.1 µM) for 4 h, and then incubated with TMRM and , 2
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examined under CLSM as described above.
Mitochondrial H O Production - 0.1 mg mitochondrial protein was added to 0.5 ml
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potassium phosphate buffer (100 mM, pH 8.0) containing 5 mM succinate. 25 µM luminol and
0.7 IU horseradish peroxidase were added, and chemiluminescence monitored continuously for
20 min at 37oC. The amount of H O produced was determined by area under the curve.
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Mitochondrial Oxygen Consumption - Mitochondrial protein (1 mg) was added to 2.0 ml
of respiration buffer (70 mM sucrose, 230 mM mannitol, 2 mM HEPES, 5 mM KH PO , 5 mM
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MgCl , 0.5 mM EDTA, pH 7.4). Oxygen consumption was measured with a Clark-type oxygen
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electrode (Hansatech Instruments, Norfolk, UK). Respiration was measured in the presence of 5
mM succinate, and state 3 respiration was initiated with the addition of 0.35 mM ADP.
Mitochondrial Membrane Potential - Mitochondrial potential was qualitatively assessed
using TMRM fluorescence intensity (ex/em = 550 nm/575 nm). Isolated mitochondria (0.3 mg)
were added to 1.5 ml buffer (70 mM sucrose, 230 mM mannitol, 3 mM HEPES, 2 mM tris-
phosphate, 5 mM succinate, 1 µM rotenone) containing TMRM (0.4 - 2 µM) and potential
assessed by quenching of the fluorescent signal.
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Mitochondrial Swelling Assays – Isolated mitochondria (0.1 mg) were added to 0.35 ml w
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buffer (70 mM sucrose, 230 mM mannitol, 3 mM HEPES, 2 mM tris-phosphate, 5 mM d
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succinate, 1 µM rotenone) and swelling was measured by decrease in absorbance at 540 nm ttp
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using a 96-well plate reader (Molecular Devices, Sunnyvale, CA). .jb
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Mitochondrial Cytochrome c Release - Isolated mitochondria (0.75 mg/2 ml) were b/
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incubated in the absence or presence of SS-02 for 100 s prior to addition of Ca2+ to induce st o
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swelling. Swelling was measured by light scattering at 610 nm. Alamethicin (7 µg/ml) was a
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added to induce maximal swelling, and the magnitude of swelling induced by Ca2+ was , 20
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expressed as a percentage of maximal swelling. After incubation for 200 s, the mitochondrial
pellet was collected by centrifugation. Cytochrome c content in the pellet and supernatant was
determined using a commercial rat/mouse cytochrome c immunoassay kit (R & D Systems,
Minneapolis, MN).
Ischemia-Reperfusion Studies – Details of the isolated perfused guinea pig heart model
have been published previously (42). Isolated hearts were perfused continuously with either
Krebs-Hensleleit solution or Krebs-Henseleit solution containing various SS peptides and
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allowed to stabilize for 30 min. Contractile force was measured with a small hook inserted into
the apex of the left ventricle and the silk ligature tightly connected to a Grass force-displacement
transducer. Global ischemia was then induced by complete interruption of coronary perfusion
for 30 min. Reperfusion was carried out for 90 min after ischemia.

RESULTS

Antioxidant Properties of SS Peptides – The antioxidant properties of SS peptides were
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demonstrated by their ability to scavenge H2O2 and inhibit the oxidation of linoleic acid and own
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LDL in vitro. The prototype peptide, SS-02, dose-dependently reduced the luminol-derived ed
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chemiluminescence produced by H2O2 in the presence of HRP (Fig. 1A). SS-02 also dose- http
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dependently inhibited the oxidation of fatty acids (Fig. 1B) and LDL in vitro (Fig. 1C). The w
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antioxidant activity of SS-02 was not dependent on the specific order of the four amino acids as brg/
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SS-31 showed similar antioxidant activity (Fig. 1D and 1E). However, substitution of Dmt1 by es
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Phe1 (SS-20) eliminated antioxidant activity (Fig. 1D and 1E). nu
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[Fig. 1]

Cellular Uptake of SS-02 – To demonstrate that the SS peptides are cell-permeable, we
incubated Caco-2 cells with [3H]SS-02 at 37oC for 60 min and measured the amount of
radioactivity in cell lysate. [3H]SS-02 was readily taken up into Caco-2 cells. The amount of
[3H]SS-02 in cell lysate and media averaged 6152 ± 128 cpm and 229622 ± 2199, respectively
(mean ± S.E.; n = 6). Based on a cell volume of ~3.3 µl/mg protein (43) and 200 µl of media,
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