Báo cáo khoa học: Microcin J25 induces the opening of the mitochondrial transition pore and cytochrome c release through superoxide generation doc

Microcin J25 induces the opening of the mitochondrial
transition pore and cytochrome c release through
superoxide generation
Marı
´
a Niklison Chirou, Augusto Bellomio, Fernando Dupuy, Beatriz Arcuri, Carlos Minahk
and Roberto Morero
Departamento de Bioquı
´
mica de la Nutricio
´
n, Instituto Superior de Investigaciones Biolo
´
gicas (Consejo Nacional de Investigaciones
Cientı
´
ficas y Te
´
cnicas—Universidad Nacional de Tucuma
´
n), Instituto de Quı
´
mica Biolo
´
gica ‘‘Dr. Bernabe Bloj,’’ San Miguel de Tucuma
´
n,
Argentina
Microcin J25 (MccJ25), a 21-amino acid antimicrobial
peptide that is active against certain human pathogens
such as Salmonella and Shigella [1], has an unusual

lasso distinctive structure [2–4] and a dual mechanism
of action. Microcin J25 inhibits transcription by
obstructing the RNA polymerase secondary channel
[5] and affects, independently, the cytoplasmic
membrane of Escherichia coli and Salmonella enterica
serovars [6]. In this regard, it was shown that MccJ25
disrupts the membrane integrity of S. enterica and
therefore causes dissipation of its membrane electrical
potential. In addition, MccJ25 inhibits respiratory
enzymes such as NADH, succinate dehydrogenase and
lactate dehydrogenase and alters the oxygen
consumption rate in vivo and in vitro [7]. The fact that
MccJ25 is a membrane-active peptide is also supported
by studies carried out on liposomes [8].
Recently, the effect of MccJ25 on intact rat heart
mitochondria was explored [9]. The peptide modifies
the membrane permeability, displays a potent effect as
inhibitor of thecomplex III and diminishes drastically
the internal ATP level. Mitochondria play a vital role
in the regulation of energy metabolism and cell death
by apoptosis and necrosis. Mitochondrial function
requires a continuous transmembrane potential, which
depends on the generation of an electrochemical pro-
ton gradient across the inner membrane. The loss of
the mitochondrial membrane integrity induces the
release of pro-apoptotic proteins [10–13]. On the one
hand, the apoptotic cascade can be triggered by a
Keywords
antibiotics; Ca
2+

; cytochrome c; microcin;
mitochondria
Correspondence
R. Morero, Chacabuco 461,
S.M. de Tucuma
´
n 4000, Argentina
Fax: +54 0381 4248025
Tel: +54 0381 4248921
E-mail:
(Received 23 April 2008, revised 21 May
2008, accepted 12 June 2008)
doi:10.1111/j.1742-4658.2008.06550.x
Microcin J25, an antimicrobial lasso-structure peptide, induces the opening
of mitochondrial permeability transition pores and the subsequent loss of
cytochrome c. The microcin J25 effect is mediated by the stimulation of
superoxide anion overproduction. An increased uptake of calcium is also
involved in this process. Additional studies with superoxide dismutase,
ascorbic acid and different specific inhibitors, such as ruthenium red, cyclo-
sporin A and Mn
2+
, allowed us to establish a time sequence of events
starting with the binding of microcin J25, followed by superoxide anion
overproduction, opening of mitochondrial permeability transition pores,
mitochondrial swelling and the concomitant leakage of cytochrome c.
Abbreviations
carboxy-DCF, 5-(-6)-carboxy-2¢,7¢-dichlorofluorescein; carboxy-H
2
DCFDA, 5-(-6)-carboxy-2¢,7¢-dichloro-dihydrofluorescein diacetate; carboxy-
H

2
DCFH, 5-(-6)-carboxy-2¢,7¢-dichloro-dihydrofluorescein; CsA, cyclosporin A; DNP, 2,4-dinitrophenol; FITC, fluorescein isothiocyanate;
MccJ25, microcin J25; MccJ25F*, fluorescent derivative of microcin J25; MTP, mitochondrial transition pore; ROS, reactive oxygen species;
RR, ruthenium red; SOD, manganese-superoxide dismutase.
4088 FEBS Journal 275 (2008) 4088–4096 ª 2008 The Authors Journal compilation ª 2008 FEBS
Ca
2+
-independent mechanism that involves the Bcl-2
protein family, but this is not necessarily associated
with mitochondrial volume changes [14]. On the other
hand, the opening of MTP could be activated by
Ca
+2
, resulting in mitochondrial swelling with the con-
sequent loss of the electrochemical gradient and the
uncoupling of oxidative phosphorylation [15]. As a
result, cytochrome c and other proteins are released
into the cytosol. The leakage of cytochrome c from
mitochondria is considered to be an early critical event
in apoptotic cascade induction, which ultimately leads
to programmed cell death [16–19]. However, additional
evidence from studies with intact cells and isolated
mitochondria suggests that mitochondrial membrane
permeability changes may also occur by some other
mechanism [20].
In the present study we analysed the effect of
MccJ25 on isolated heart mitochondria. Our results
indicated that MccJ25 induces the overproduction of
superoxide anions, thus increasing the mitochondrial
inner membrane permeability and activation of the

mitochondrial transition pore (MTP), resulting in
swelling and cytochrome c release.
Results
Mitochondrial uptake of MccJ25
Mitochondrial uptake of MccJ25 was examined using
the peptide fluorescent derivative MccJ25F*, which
showed antibiotic activity and membrane gradient dis-
sipater capability similar to the native peptide (data
not shown). Addition of MccJ25F* to energized mito-
chondria (in the presence of 10 mm succinate) resulted
in the immediate uptake of the microcin analogue
(Fig. 1). The uptake was rapid, with a maximal level
reached within 20 min. To ensure that the uptake of
MccJ25F* was not an artifact, we confirmed the
results by determining binding displacement with
native MccJ25 (see Fig. 1) and fluorescence in the
mitochondrial pellet (results not shown). Studies car-
ried out with nonenergized mitochondria (i.e. in the
absence of succinate) showed a marked decrease in the
uptake capability of MccJ25F
*
. Pretreatment of ener-
gized mitochondria with either 200 lm vanadate (an
ATPase inhibitor) or 100 lm 2,4-dinitrophenol
(2,-DNP; anelectrochemical gradient dissipater)
reduced the uptake of MccJ25F* by approximately
85%, suggesting that the uptake of MccJ25F* depends
on both the energy of mitochondria and the
mitochondrial proton membrane gradient, mainly
determined by ATP level and provided by succinate

oxidation, respectively.
Effect of MccJ25 on the mitochondrial transition
pore of energized mitochondria
The addition of 20 lm MccJ25 induced the swelling of
energized mitochondria (Fig. 2). The kinetics of swell-
ing during a period of 5 h had a linear pattern charac-
teristic of MTP induction, showing an initial rate of
7.02 4 light scattering ⁄ min · 10
4
. The initial rate of
swelling increased as a function of MccJ25 concentra-
tion (inset in Fig. 2) and became saturated at about
60 lm (results not shown). Similar results were
obtained, although with reduced swelling, when the
buffer was devoid of succinate. Furthermore, the pres-
ence of 10 lm antimycin A inhibited completely the
swelling, suggesting that the electronic flow through
the respiratory chain stimulates the MccJ25 effect
(results not shown). We next determined whether
MccJ25 was able to induce the permeation of solutes
with a relatively low molecular mass. To study this we
loaded mitochondria with calcein-AM, which is con-
verted into calcein by endogenous esterases. Loaded
mitochondria were treated with increasing concentra-
tions of MccJ25 and the release of calcein was studied.
A marked calcein release, parallel to the increased
swelling, was observed following the addition of 20 lm
MccJ25 (Fig. 2).
0
5

10
15
20
25
30
35
Uptake (%)
Time (min)
0 5 10 15 20 25 30 35
Fig. 1. Uptake of MccJ25 by isolated mitochondria. Energized
mitochondria without pre-incubation (
) or pre-incubated with
200 l
M vanadate ( ), 100 lM 2,4-DNP (d)or5lM native MccJ25
(h), were suspended in 10 m
M Tris–sodium phosphate buffer
(pH 7.4), 230 m
M mannitol, 70 mM sucrose, 3 mM HEPES, supple-
mented with 10 m
M succinate and 1 lM rotenone. Nonenergized
mitochondria (.) were suspended in the same buffer but in the
absence of succinate. Then, 1.4 l
M MccJ25F* (final concentration)
was added to the suspensions, which were incubated for different
periods of time at 25 °C. The fluorescence incorporated by mito-
chondria was plotted as a function of the incubation time. Results
are expressed as mean ± SD of five independent experiments.
M. Niklison Chirou et al. MccJ25 induces the opening of mitochondrial transition pore
FEBS Journal 275 (2008) 4088–4096 ª 2008 The Authors Journal compilation ª 2008 FEBS 4089
In an attempt to identify the role played by the

MTP in the swelling and permeability changes of mito-
chondria induced by MccJ25, we tested the influence
of the specific pore inhibitor cyclosporin A (CsA), a
calcium chelator (EDTA), a calcium competitor
(Mn
2+
) and the calcium uniporter inhibitor ruthenium
red (RR). As shown in Table 1, swelling and calcein
release induced by MccJ25 were almost completely
inhibited by 1 lm CsA. Additionally, 50 lm EDTA,
125 lm Mn
2+
and 2.5 lm RR also almost completely
inhibited the swelling induced by MccJ25, strongly
suggesting that the peptide was able to induce mito-
chondrial swelling through the opening of the MTP
mediated by the uptake of calcium through the uniport
of calcium. On the other hand, KCN and antimycin A,
both inhibitors of electron transport, also inhibited the
swelling induced by MccJ25. The inhibition of swelling
induced by ascorbic acid will be discussed later. As a
positive control, the swelling and calcein release
induced by calcium, and the inhibitory effect of CsA
and RR, are also shown in Table 1, confirming previ-
ous results [21,22].
Cytochrome c release
An initial aim of this work was to study the influence
of MccJ25 on the release of cytochrome c. Therefore,
we investigated whether the indirect opening of MTP
by MccJ25 was followed by the release of cyto-

chrome c. The experimental results (inset of Fig. 3)
showed that increasing amounts of cytochrome c were
released, showing a positive linear correlation with the
0 50 100 150 250200 300
–0.4
–0.3
–0.2
–0.1
0.0
0.1
0 10203040
0
2
4
6
8
10
12
14
2
4
Light scattering (540 nm)
Time (min)
Δ
Light scattering
·min
–1
x 10
4
MccJ25 (µM)

Fluorescence (AU)
Fig. 2. Effect of MccJ25 on mitochondrial swelling. The time
course of swelling (h) and calcein release (D) of energized mito-
chondria induced by 20 l
M MccJ25 was assessed by measuring
the change of light scattering at 540 nm or of calcein fluorescence,
respectively, of mitochondria suspended in buffer, as described in
the Materials and methods. Controls of swelling (
) and calcein
release (
) were performed by incubation in the absence of
MccJ25. The data shown are representative of at least five sepa-
rate studies. The inset shows mitochondrial swelling as a function
of MccJ25 concentration.
Table 1. Effect of different drugs on the swelling and calcein
release from mitochondria induced by MccJ25. Swelling and calcein
release of energized mitochondria were induced by 20 l
M MccJ25
and 50 l
M Ca
+2
in the absence or presence of different inhibitors.
ND, not determined.
Mitochondrial
swelling DLight
scattering ⁄
min · 10
4
Calcein
release (%)

MccJ25 (20 l
M) 7.02 ± 0.01
a
52.00 ± 1.00
+EDTA (50 l
M) 1.19 ± 0.01 ND
+Mn
2+
(125 lM) 0.01 ± 0.02 0.10 ± 0.10
+RR (2.5 l
M) 0.28 ± 0.01 ND
+CsA (1 l
M) 0.30 ± 0.01 4.80 ± 1.00
+Ascorbic acid (0.5 m
M) 0.09 ± 0.02 ND
+Antimycin A (10 l
M) 0.50 ± 0.02 ND
+KCN (2 m
M) 0.43 ± 0.10 ND
Calcium (50 l
M) 15.20 ± 0.02 54.50 ± 2.00
+CsA (1 l
M) 0.10 ± 0.01 0.30 ± 0.01
+RR (2.5 l
M) 0.25 ± 0.01 ND
Control 0.03 ± 0.01 0.30 ± 0.10
a
Results are expressed as means ± SD of three separate
experiments.
0 50 100 150 200

0
4
8
12
16
0 10203040
0
2
4
6
8
10
12
Cyt c release (pmol·mg
–1
protein)
Cyt c release (pmol·mg
–1
protein)
Time (min)
MccJ25 (µM)
Fig. 3. Effect of MccJ25 on cytochrome c release from mitochon-
dria. Isolated mitochondria were suspended in 100 m
M potassium
phosphate buffer (pH 7.4), 10 m
M succinate, 1 lM rotenone, and
incubated for different periods of time at 37 °C in the absence (h)
and in the presence of 20 l
M MccJ25 ( ). Cytochrome c was
determined chromatographically, as described in the Materials and

methods. Data are the mean ± SD of three independent experi-
ments. The inset shows cytochrome c release as a function of the
MccJ25 concentration after 2 h of incubation at 37 °C.
MccJ25 induces the opening of mitochondrial transition pore M. Niklison Chirou et al.
4090 FEBS Journal 275 (2008) 4088–4096 ª 2008 The Authors Journal compilation ª 2008 FEBS
concentration of MccJ25. This result agrees with the
swelling effect induced by MccJ25. It should be noted
that at 20 lm MccJ25 the release effect was time
dependent. After a lag phase of approximately 50 min,
the release of cytochrome c increased abruptly, reach-
ing a plateau at about 3 h (Fig. 3).
Effect of MccJ25 on the production rate of
superoxide anions
It was recently found in our laboratory that the effect
of MccJ25 on the E. coli respiratory chain enzymes is
mediated by increased superoxide production [6]. As it
is widely accepted that the mitochondrial electron
transport chain is the main source of superoxide
anions, we decided to determine the effect of MccJ25
on the mitochondrial electron transport chain and the
possible implications on the swelling and permeability
changes. As shown in Fig. 4, the rate of O
À
2
generation
by the submitochondrial particles increased as a func-
tion of time, reaching a plateau at just about 45 min
(data not shown in Fig. 4). The effect was dependent
on the MccJ25 concentration (inset of Fig. 4) and was
almost completely inhibited by superoxide dismutase

(SOD), ensuring the superoxide anion determination.
To demonstrate and better understand the mechanism
of the reactive oxygen species (ROS) overproduction
induced by the antimicrobial peptide, MccJ25, we
examined the effect on isolated mitochondria. The
ROS production was monitored using a ROS-sensitive
fluorescent probe [5-(-6)-carboxy-2¢,7¢-dichloro-dihy-
drofluorescein diacetate (carboxy-H
2
DCFDA)]. The
results shown in Fig. 5 indicate that 20 lm MccJ25
induced a clear increment of ROS production com-
pared with the control experiment performed in the
absence of the peptide. Indeed, the rate of ROS pro-
duction in the presence of MccJ25 was three times
higher than in the absence of this peptide. The maxi-
mum ROS production rate was obtained after 30 min
of exposure to antibiotic. Pretreatment of isolated
mitochondria with antioxidants such as 0.5 mm ascor-
bic acid or 0.5 mm a-tocoferol acetate almost com-
pletely suppressed the ROS production induced by
MccJ25. By contrast, pretreatment with 2.5 lm RR
was unable to prevent the ROS overproduction
induced by MccJ25.
Effect of MccJ25 on NADPH oxidation
Reactive oxygen species produced in mitochondria are
inactivated by a set of protective enzymes, including
SOD, glutathione peroxidase and glutathione reductase
[23]. Glutathione reductase recycles oxidized glutathi-
one to its reduced form, using electrons from the

NADPH. As a consequence, the overproduction of
superoxide anions induced by MccJ25 would implicate
0 5 10 20 3015 25
0.0
0.2
0.4
0.6
0.8
1.0
1.2
0 102030405060
0.0
0.4
0.8
1.2
O
2
–
Production (nmoles)
O
2
–
Production (nmoles)
Time (min)
MccJ25 (µM)
Fig. 4. Rate of O
À
2
generation induced by MccJ25 in rat heart sub-
mitochondrial particles. The rate of superoxide generation in the

presence of 20 l
M MccJ25 (d) or in the presence of 20 lM
MccJ25 and manganese-SOD (300 unitsÆmL
)1
)( ) was determined
as described in the Materials and methods. Data are mean ± SD of
three independent experiments. The rate of superoxide generation
as a function of MccJ25 concentration is shown in the inset.
0
500
1000
1500
2000
2500
Fluorescence (AU)
0 5 10 15 20 25 30 35
Time (min)
Fig. 5. Effects of MccJ25 on the generation of ROS in isolated
mitochondria. A preparation of mitochondria, preloaded with the flu-
orescence probe carboxy-H
2
DCFDA, was suspended in 10 mM
Tris–potassium phosphate buffer (pH 7.4), 150 mM sucrose, 50 mM
KCl, 1 lM rotenone (h), or in buffer containing 20 lM MccJ25 ( ).
Pretreatment with 2.5 l
M RR (s), 0.5 mM a-tocopherol acetate (d)
or 0.5 m
M ascorbic acid (Ñ) was also performed. The time course
of 5-(-6)-carboxy-2¢,7¢-dichlorofluorescein (carboxy-DCF) fluores-
cence (k

ex
, 485 nm; k
em
, 525 nm) was monitored after the addition
of 10 m
M succinate. The data shown are representative of at least
five separate studies.
M. Niklison Chirou et al. MccJ25 induces the opening of mitochondrial transition pore
FEBS Journal 275 (2008) 4088–4096 ª 2008 The Authors Journal compilation ª 2008 FEBS 4091
oxidation of NADPH. As shown in Fig. 6, 20 lm
MccJ25 markedly increased the oxidation of NADPH
compared with a control in the absence of the peptide.
EDTA, RR and ascorbic acid completely inhibited the
effect of MccJ25. Surprisingly, CsA, a specific inhibitor
of the pore, was unable to inhibit the effect of MccJ25,
which strengthens the hypothesis that MccJ25 gener-
ates ROS, regardless of the presence of calcium.
Discussion
Understanding the mechanisms of action of some types
of proteins and peptides on the structural and func-
tional state of mitochondria seems to be important in
the development of apoptosis-regulating technologies,
particularly for anticancer therapy, and for the design
of new classes of antibiotics, taking into account a cer-
tain similarity between mitochondria and bacteria. In
this work we observed that mitochondrial uptake of
MccJ25 proceeds very rapidly and in a concentration-
dependent manner. The peptide inserts into the mem-
brane, modifying its permeability and provoking conse-
quently an electrical potential dissipation, as described

previously [9]. Such an effect promotes the uncoupling
of electron transport with a concomitant loss of the
efficiency to reduce oxygen and an increase in superox-
ide species and subsequently ROS generation. Interest-
ingly, this MccJ25 effect is similar to that described
recently for E. coli [6]. The membrane perturbations
would induce a small increase in the internal calcium
concentration, which in turn activates the uniporter of
calcium, increasing even more the calcium influx and
ROS production. Both the increase of intramitochond-
rial calcium concentration and the opening of MTP
trigger mitochondrial swelling, with the concomitant
release of the apoptotic inducer cytochrome c [24]. The
sequence of these events is schematically represented in
Fig. 7 and ordered on a timescale according to the
0 20 40 60 80 100
–40
–35
–30
–25
–20
–15
–10
–5
0
5
Fluorescence (AU)
Time (min)
Fig. 6. Effect of MccJ25 on the mitochondrial NADPH level. Mito-
chondria suspended (0.5 mgÆmL

)1
) in Tris–potassium phosphate
buffer (pH 7.4), 10 m
M succinate, 1 lM rotenone, 150 mM sucrose,
50 m
M KCl were incubated in the absence (d) or in the presence
of 20 l
M MccJ25 without any pretreatment (D), or pretreated for
1 min with 1 l
M CsA ( ), 2.5 lM RR (s), 0.1 mM EDTA (.)or
0.5 m
M ascorbic acid (h). At different time-points NADPH intrinsic
fluorescence was monitored at excitation and emission wave-
lengths of 366 and 450 nm respectively.
60 min
Cyt.
r
c
elease
Seg
15 min
45 min
8 min
5 min
MccJ25
Fluidity
increase
Gradient
dissipation
Uncoupling

O
2
–
production
ROS
increase
SOD
Vit. C
Vit. E
NADPH
Ca
2+
Calcium
uniporter
MTP
opening
Swelling
RR
EDTA
CsA
ADP, Mn
2+
Timescale events
Uptake
Fig. 7. Schematic representation of the time sequence steps triggered by MccJ25 acting on isolated heart mitochondria.
MccJ25 induces the opening of mitochondrial transition pore M. Niklison Chirou et al.
4092 FEBS Journal 275 (2008) 4088–4096 ª 2008 The Authors Journal compilation ª 2008 FEBS
kinetics of each process. The interaction between
MccJ25 and the membrane is followed by membrane
perturbation, superoxide and ROS overproduction,

stimulation of the calcium uniporter, opening of the
MTP and, finally, mitochondrial swelling with cyto-
chrome c leakage. This sequence is supported by the
effect of several specific inhibitors. The overproduction
of superoxide and ROS is the first event induced by
MccJ25 and is responsible for triggering the subsequent
effects because the presence of ascorbic acid was suffi-
cient to inhibit mitochondrial swelling (see Table 1).
The kinetic of superoxide and ROS overproduction
clearly demonstrates that the generation of this reactive
species precedes MTP opening and mitochondrial
swelling. The inability of RR and CsA, specific inhibi-
tors of the calcium uniport and MTP respectively, to
inhibit the increase of ROS strongly supports the
above-mentioned hypothesis. The NADPH oxidation
occurs prior to the opening of the MTP pore and is
coupled to the transport of calcium from the intermem-
brane space to the matrix because it is inhibited by RR
but not by CsA. The mitochondrial swelling induced by
MccJ25 was also inhibited by EDTA, Mn
2+
and RR,
indicating that the influx of Ca
2+
to the mitochondrial
matrix was necessary for activation of the MTP. These
results indicate that the effect of MccJ25 on the respira-
tory chain forces the opening of the MTP pore, which
is mediated by an increase of the matrix calcium con-
centration, with the concomitant release of cyto-

chrome c.
The fact that the mitochondrial swelling induced by
MccJ25 was inhibited by antimycin A allowed us to
presume that the peptide effect takes place only when
the electron flow through the respiratory chain is oper-
ating. In addition, and supporting this result, the swell-
ing effect is highly elevated in activated mitochondria
and completely inhibited by rotenone (complex I inhib-
itor) in ‘not activated’ mitochondria. Alternatively, we
could consider that in the presence of antimycin A the
mitochondrial membrane potential cannot be built
up and therefore mitochondria would not accumulate
calcium, which is required for MTP induction.
In conclusion, we have shown that MccJ25 has a
mitochondrial deleterious effect that is associated with
the induction of the MTP. Microcin J25 would target
the site of anion superoxide generation, increasing the
production of ROS. However, the results presented
here do not clarify the mechanism by which MccJ25
induces ROS production. Superoxide can be produced
at complex I and ⁄ or at complex III [25]. Our results
clearly indicate that complex III is essentially impli-
cated in the mechanism of superoxide production
because the peptide effect was obtained in the presence
of rotenone, a specific inhibitor of complex I. We dem-
onstrated that ROS play a major role in mediating
mitochondrial dysfunction induced by MccJ25. Taking
into account the induced swelling and the cytochrome
c release, we could hypothesize that MccJ25 behaves
as an apoptotic agent. However, any disruption in the

electrochemical gradient and ⁄ or oxidative phosphory-
lation, resulting in a decrease of ATP production,
could compromise the progression of this form of cell
death because an energy requirement is clearly needed
for apoptosome formation. Moreover, there is contro-
versy about whether or not the mitochondria indeed
swell during apoptosis. Some studies have reported
observing mitochondrial swelling [26], whereas others
have reported that swelling never occurred [27,28].
Recent investigations suggest that cytochrome c release
in apoptosis was not caused by mitochondrial swelling
[29]. Additional studies will be helpful to understand,
in greater detail, the molecular basis of this effect, its
biological significance and the possible peptide
bio-applicability mainly in whole human cells. These
studies are currently underway in our laboratory.
Materials and methods
Chemicals and reagents
Calcein-AM, CsA, fluorescein isothiocyanate (FITC) and
ATP were purchased from Sigma Chemical Co. (St Louis,
MO, USA). Carboxy-H
2
DCFDA-SE was obtained from
Molecular Probes Inc. (Eugene, OR, USA). All other
reagents were of analytical grade or the purest available
commercial form.
Peptides synthesis and purification
Microcin J25 was obtained from the supernatant of E. coli
AB259 harboring pTUC200 and was purified according to
the procedure previously reported [7]. This procedure

yielded a preparation that appeared homogeneous in two
different systems of analytical RP-HPLC [30,31]. A fluores-
cent analog containing FITC (MccJ25F*) was prepared for
mitochondrial uptake studies. A mutated peptide in which
isoleucine of position thirteen was replaced with lysine was
obtained and purified from the supernatant of E. coli
DH5a harboring pI13K. The strain was generously
provided by P. Vincent (INSIBIO, CONICET ⁄ UNT,
S. M. Tucuma
´
n). The fluorescent peptide was obtained by
incubation of the mutated peptide with FITC (1 : 3, w ⁄ w)
for 2 h in alkaline medium and in darkness at room tem-
perature (25 °C). The labeled peptide was purified by chro-
matography on a hydrophobic C8 cartridge. The fraction
eluted with 100% methanol was reduced under vacuum,
resuspended in water and chromatographed on a C18
M. Niklison Chirou et al. MccJ25 induces the opening of mitochondrial transition pore
FEBS Journal 275 (2008) 4088–4096 ª 2008 The Authors Journal compilation ª 2008 FEBS 4093
column using a Gilson HPLC system, being finally eluted
with a 50 mm sodium phosphate buffer (pH 6.5) ⁄ methanol
gradient. Peptide concentrations were determined by mea-
surement of the absorbance at 278 nm [32].
Isolation of heart mitochondrial
Winstar rats (250–300 g) were killed by CO
2
inhalation, in
accordance with the European directive for protection of
vertebrate animals for scientific research. Hearts were rap-
idly removed and placed in 10 mL of ice-cold 5 mm

HEPES buffer (pH 7.4), 200 mm mannitol, 10 m m sucrose,
1mm EDTA, 0.1% BSA. The tissue was finely minced with
scissors and then homogenized using an Omni-Mixer (Sor-
vall, Norwalk, CT, USA). The homogenate was centrifuged
at 900 · g for 10 min, the pellet was discarded and the
supernatant centrifuged again at 17 000 · g for 10 min.
The pellet containing the mitochondria was resuspended in
the isolation buffer without EDTA. To prepare submito-
chondrial particles, the mitochondrial pellet was resus-
pended (20 mgÆmL
)1
)in50mm Tris–HCl (pH 7.6), 230 mm
mannitol, 70 mm sucrose, and sonicated three times (each
consisting of a 30-s pulse burst) at 1-min intervals, at 4 °C.
The sonicated mitochondria were centrifuged at 8500 · g
for 10 min to remove the unbroken organelles. The super-
natant was centrifuged again at 100 000 · g for 60 min,
and the resulting pellet was washed and resuspended in the
same buffer [33]. The protein concentration was determined
by the method of Lowry et al., with bovine albumin as the
standard [34].
Mitochondrial uptake studies
For mitochondrial uptake of MccJ25F*, mitochondria
(1 mgÆmL
)1
) were suspended in 10 mm Tris–sodium phos-
phate buffer (pH 7.4), 230 mm mannitol, 70 mm sucrose,
3mm HEPES, supplemented with 10 mm succinate and
1 lm rotenone, at 25 °C, then 1.4 lm MccJ25F* was added
and the solution was incubated at 25 °C. Uptake was

stopped at different time-points by centrifugation
(12 000 · g, 5 min, 4 °C) and fluorescence of tyrosine (k
ex
:
277 nm, k
em
: 305 nm) and FITC (k
ex
: 490 nm, k
em
:
520 nm) in the supernatant was measured using an ISS
(Champaign, IL, USA) PC1 spectrofluorometer at 25 °C.
Uptake experiments were also determined in mitochondrial
samples incubated previously with 200 mm vanadate, or
100 mm DNP, for 5 min at 37 °C.
Mitochondrial swelling assay
Isolated mitochondria (1 mgÆmL
)1
) were incubated in
10 mm Tris–sodium phosphate buffer (pH 7.4), 230 mm
mannitol, 70 mm sucrose, 3 mm HEPES, supplemented
with 10 mm succinate and 1 lm rotenone at 25 °C [27].
Different quantities of MccJ25 were added to the incuba-
tion buffer. In additional experiments, inhibitors such as
EDTA, Mn
2+
, CsA, KCN, antimycin A, ascorbic acid and
RR were added before the addition of MccJ25. Calcium
(50 lm) was used as a positive control. A control

experiment in the absence of MccJ25 and Ca
2+
was also
performed. Swelling was estimated from the changes of
light scattering at 540 nm in a DU7500 spectrophotometer
(Beckman, Fullerton, CA, USA) equipped with a peltier
constant temperature chamber. The rate of swelling (Dlight
scattering ⁄ min) was calculated from the slope of the initial
linear portion of the curve.
Calcein release from mitochondria
Mitochondria isolated from rat heart were incubated at
25 °C for 30 min in the suspension buffer containing 2 lm
calcein-AM and then washed and resuspended in the same
buffer. To assess calcein release, loaded mitochondria
(0.1 mgÆmL
)1
) were added to the assay buffer [5 mm Tris–
HEPES buffer (pH 7.4), 250 mm sucrose, 0.1% BSA,
10 lm CoCl
2
] and variations in the fluorescence were mea-
sured using an ISS PC1 spectrofluorometer [22]. The excita-
tion and emission wavelengths were 488 and 530 nm
respectively. The buffer contained 10 mm CoCl
2
to quench
the fluorescence of calcein released from mitochondria.
The fluorescence value obtained in the presence of 0.2%
Triton X-100 was considered as 100% leakage.
Cytochrome c release

Quantification of cytochrome c release from rat heart mito-
chondria was performed as described by Crouser et al. [24],
with minor modifications. Essentially, intact mitochondria
(1 mgÆmL
)1
) suspended in 100 mm potassium phosphate
buffer (pH 7.4), 10 mm succinate and 1 lm rotenone were
incubated at 37 °C with MccJ25. At different time-points,
aliquots were centrifuged (30 000 g, 10 min, 4 °C) and the
supernatant was analyzed using HPLC (Gilson HPLC
equipped with a UV-VIS detector) through a C8 reverse-
phase analytical column (Waters XTerra MS C8 5 l m,
4.6 · 250 mm) preceded by a guard column. A linear gradi-
ent, increasing from 20 to 60% acetonitrile in water, was
employed. Both the 20% and the 60% acetonitrile solutions
also contained 100 mm KCl and 0.1% trifluoroacetic acid
(v ⁄ v). The eluted cytochrome c was detected at 393 nm.
Cytochrome c concentrations were calculated from a
standard measurement.
Superoxide anion radical generation
The rate of O
À
2
generation by submitochondrial particles
was measured as reduction of acetylated ferricytochrome c,
which is an excellent quantitative trap for O
À
2
[35]. The
MccJ25 induces the opening of mitochondrial transition pore M. Niklison Chirou et al.

4094 FEBS Journal 275 (2008) 4088–4096 ª 2008 The Authors Journal compilation ª 2008 FEBS
reduction was followed spectrophotometrically at 550 nm
in a Beckman DU 7500 at 25 °C. The reaction mixture
contained 100 mm potassium phosphate buffer (pH 7.4),
10 lm acetylated ferricytochrome c,1lm rotenone, small
mitochondrial particles (0.5 mg of proteinÆmL
)1
) and
20 lm MccJ25. The reaction was started by the addition of
10 mm succinate. We also performed experiments in which
the effect of MccJ25 was studied after pre-incubation for
5 min with SOD.
Reactive oxygen species production
Reactive oxygen species production was monitored using
the ROS-sensitive fluorescent probe, 5-(-6)-carboxy-2¢,
7¢-dichloro-dihydrofluorescein diacetate (carboxy-H
2
DCFDA).
Once in the mitochondria, the acetate groups are cleaved
by nonspecific esterases, hence the nonfluorescent 5-(-6)-
carboxy-2¢,7¢-dichloro-dihydrofluorescein (carboxy-H
2
DCFH)
is trapped inside. This probe was selected because is well
retained not only in cells but also in mitochondria [36].
Oxidation of carboxy-H
2
DCFH by ROS yields carboxy-
DCF. This fluorescent product indirectly measures the O
À

2
produced that has dismutated to H
2
O
2
through the action
of endogenous Mn
2+
-dependent SOD. The suspension of
mitochondria (10 mgÆmL
)1
) was incubated with carboxy-
H
2
DCFDA for 30 min at 30 °C and then washed twice
with 10 mm Tris–sodium phosphate buffer (pH 7.4),
150 mm sucrose and 50 mm KCl to eliminate nonincorpo-
rated probe. To monitor ROS production, carboxy-
H
2
DCFH-loaded mitochondria were suspended
(0.1 mgÆmL
)1
)in10mm Tris–sodium phosphate buffer
(pH 7.4), 150 mm sucrose, 50 mm KCl, 10 mm succinate,
1 lm rotenone, and the variation in fluorescence was
followed using an ISS PC1 spectrofluorometer at 25 °C
after the addition of MccJ25. The excitation and emission
wavelengths were 490 and 520 nm respectively.
Determination of NADPH oxidation

Mitochondrial pyridine nucleotides were monitored, in an
ISS PC1 spectrofluorometer at 25 °C, by measuring their
intrinsic fluorescence at 450 nm after exciting at 340 nm
[37]. Mitochondria were suspended (0.1 mgÆmL
)1
)in10mm
Tris–potassium phosphate buffer (pH 7.4), 10 mm succi-
nate, 1 lm rotenone, 150 mm sucrose and 50 mm KCl. The
suspension was pre-incubated for 1 min, and the oxidation
of NADPH was started by adding 20 lm MccJ25 or 50 lm
Ca
2+
.
Acknowledgements
Financial support was provided by CONICET (Grant
PIP 4996) and CIUNT (Grant 26 ⁄ D228) and the
Agencia Nacional de Promocio
´
n Cientı
´
fica y Te
´
cnica
(PICTO 843, PAE 22642). M. V. N. and F. D. are
recipient of a CONICET fellowship. R. D. M., C. M.
and A. B. are CONICET career investigator. We
thank Monica Delgado for his generous assistance.
References
1 Salomon RA & Farias RN (1992) Microcin 25, a novel
antimicrobial peptide produced by Escherichia coli .

J Bacteriol 174, 7428–7435.
2 Bayro MJ, Swapna GV, Huang JY, Ma LC, Sineva E,
Dawson PE, Montelione GT & Ebright RH (2003)
Structure of antibacterial peptide Microcin J25:
a 21-residue lariat protoknot. J Am Chem Soc 125,
12382–12383.
3 Rosengren KJ, Clark RJ, Daly NL, Goransson U,
Jones A & Craik DJ (2003) Microcin J25 has a
threaded sidechain-to-backbone ring structure and not a
head-to-tail cyclized backbone. J Am Chem Soc 125,
12464–12474.
4 Wilson KA, Kalkum M, Ottesen J, Yuzenkova J,
Chait BT, Landick R, Muir T, Severinov K & Darst
SA (2003) Structure of microcin J25, a peptide inhibitor
of bacterial RNA polymerase, is a lassoed tail. JAm
Chem Soc 125, 12475–12483.
5 Adelman K, Yuzenkova J, La Porta A, Zenkin N,
Lee J, Lis JT, Borukhov S, Wang MD & Severinov K
(2004) Molecular mechanism of transcription inhibition
by peptide antibiotic microcin J25. Mol Cell 14, 753–
762.
6 Bellomio A, Vincent PA, de Arcuri BF, Farias RN &
Morero RD (2007) Microcin J25 has dual and indepen-
dent mechanisms of action in Escherichia coli: RNA
polymerase inhibition and increased superoxide produc-
tion. J Bacteriol 189, 4180–4186.
7 Rintoul MR, de Arcuri BF, Salomon RA, Farias RN &
Morero RD (2001) The antibacterial action of microcin
J25: evidence for disruptionof cytoplasmic membrane
energization in Salmonella newport. FEMS Microbiol

Lett 204, 265–270.
8 Rintoul MR, de Arcuri BF & Morero RD (2000)
Effects of the antibiotic peptide microcin J25 on lipo-
somes: role of acyl chain length and negatively charged
phospholipid. Biochim Biophys Acta 1509, 65–72.
9 Niklison Chirou MV, Minahk CJ & Morero RD (2004)
Antimitochondrial activity displayed by the antimicro-
bial peptide microcin J25. Biochem Biophys Res Com-
mun 317, 882–886.
10 Du C, Fang M, Li Y, Li L & Wang X (2000) Smac,
a mitochondrial protein that promotes cytochrome
c-dependent caspase activation by eliminating IAP
inhibition. Cell Death Differ 102, 33–42.
11 Kohler C, Gahm A, Noma T, Nakazawa A, Orrenius S
& Zhivotovsky B (1999) Release of adenylate kinase 2
M. Niklison Chirou et al. MccJ25 induces the opening of mitochondrial transition pore
FEBS Journal 275 (2008) 4088–4096 ª 2008 The Authors Journal compilation ª 2008 FEBS 4095
from the mitochondrial intermembrane space during
apoptosis. FEBS Lett 447, 10–12.
12 Loeffler M & Kroemer G (2000) The mitochondrion in
cell death control: certainties and incognita. Exp Cell
Res 256, 19–26.
13 Verhagen AM, Ekert PG, Pakusch M, Silke J, Connolly
LM, Reid GE, Moritz RL, Simpson RJ & Vaux DL
(2000) Identification of DIABLO, a mammalian protein
that promotes apoptosis by binding to and antagonizing
IAP proteins. Cell 102, 43–53.
14 Ott M, Robertson JD, Gogvadze V, Zhivotovsky B &
Orrenius S (2002) Cytochrome c release from mitochon-
dria proceeds by a two-step process. Proc Natl Acad Sci

USA 99, 1259–1263.
15 Antonsson B, Montessuit S, Lauper S, Eskes R &
Martinou JC (2000) Bax oligomerization is required for
channel-forming activity in liposomes and to trigger
cytochrome c release from mitochondria. Biochem J
345 (Pt 2), 271–278.
16 Bernardi P, Krauskopf A, Basso E, Petronilli V,
Blachly-Dyson E, Di Lisa F & Forte MA (2006) The
mitochondrial permeability transition from in vitro
artifact to disease target. FEBS J 273, 2077–2099.
17 Bernardi P, Scorrano L, Colonna R, Petronilli V &
Di Lisa F (1999) Mitochondria and cell death. Mecha-
nistic aspects and methodological issues. Eur J Biochem
264, 687–701.
18 Crompton M (1999) The mitochondrial permeability
transition pore and its role in cell death. Biochem J
341 (Pt 2), 233–249.
19 Zoratti M & Szabo I (1995) The mitochondrial
permeability transition. Biochim Biophys Acta 1241,
139–176.
20 Armstrong JS, Yang H, Duan W & Whiteman M
(2004) Cytochrome bc(1) regulates the mitochondrial
permeability transition by two distinct pathways. J Biol
Chem 279, 50420–50428.
21 Lee WK, Spielmann M, Bork U & Thevenod F (2005)
Cd
2+
-induced swelling-contraction dynamics in isolated
kidney cortex mitochondria: role of Ca
2+

uniporter, K
+
cycling, and protonmotive force. Am J Physiol Cell
Physiol 289, C656–C664.
22 Risso A, Braidot E, Sordano MC, Vianello A, Macri F,
Skerlavaj B, Zanetti M, Gennaro R & Bernardi P
(2002) BMAP-28, an antibiotic peptide of innate immu-
nity, induces cell death through opening of the mito-
chondrial permeability transition pore. Mol Cell Biol
22, 1926–1935.
23 Navarro A & Boveris A (2007) The mitochondrial
energy transduction system and the aging process.
Am J Physiol Cell Physiol 292, C670–C686.
24 Crouser ED, Gadd ME, Julian MW, Huff JE,
Broekemeier KM, Robbins KA & Pfeiffer DR (2003)
Quantitation of cytochrome c release from rat liver
mitochondria. Anal Biochem 317, 67–75.
25 Turrens JF & Boveris A (1980) Generation of super-
oxide anion by the NADH dehydrogenase of bovine
heart mitochondria. Biochem J 191, 421–427.
26 Narita M, Shimizu S, Ito T, Chittenden T, Lutz RJ,
Matsuda H & Tsujimoto Y (1998) Bax interacts with
the permeability transition pore to induce permeability
transition and cytochrome c release in isolated mito-
chondria. Proc Natl Acad Sci USA 95, 14681–14686.
27 Kluck RM, Esposti MD, Perkins G, Renken C, Kuw-
ana T, Bossy-Wetzel E, Goldberg M, Allen T, Barber
MJ, Green DR et al. (1999) The pro-apoptotic proteins,
Bid and Bax, cause a limited permeabilization of the
mitochondrial outer membrane that is enhanced by

cytosol. J Cell Biol 147, 809–822.
28 Von Ahsen O, Renken C, Perkins G, Kluck RM,
Bossy-Wetzel E & Newmeyer DD (2000) Preservation of
mitochondrial structure and function after Bid- or Bax-
mediated cytochrome c release. J Cell Biol 150, 1027–1036.
29 Gao W, Pu Y, Luo KQ & Chang DC (2001) Temporal
relationship between cytochrome c release and mito-
chondrial swelling during UV-induced apoptosis in
living HeLa cells. J Cell Sci 114, 2855–2862.
30 Bellomio A, Rintoul MR & Morero RD (2003) Chemi-
cal modification of microcin J25 with diethylpyrocar-
bonate and carbodiimide: evidence for essential histidyl
and carboxyl residues. Biochem Biophys Res Commun
303, 458–462.
31 Blond A, Peduzzi J, Goulard C, Chiuchiolo MJ,
Barthelemy M, Prigent Y, Salomon RA, Farias RN,
Moreno F & Rebuffat S (1999) The cyclic structure of
microcin J25, a 21-residue peptide antibiotic from
Escherichia coli. Eur J Biochem 259, 747–755.
32 Vishwanath VA & McIntosh JM (2006) Synthesis of
fluorescent analogs of -conotoxin MII. Bioconjug Chem
17, 1612–1617.
33 Boveris A (1984) Determination of the Production of
Superoxide Radicals and Hydrogen Peroxide in Mito-
chondria. Methods of Enzymatic Analysis. pp. 429–435,
Academic Press, New York, NY.
34 Lowry OH, Rosebrough NJ, Farr AL & Randall RJ
(1951) Protein measurement with the Folin phenol
reagent. J Biol Chem 193, 265–275.
35 Azzi A, Montecucco C & Richter C (1975) The use of

acetylated ferricytochrome c for the detection of super-
oxide radicals produced in biological membranes.
Biochem Biophys Res Commun 65, 597–603.
36 Xie Z, Kometiani P, Liu J, Li J, Shapiro JI & Askari A
(1999) Intracellular reactive oxygen species mediate the
linkage of Na
+
⁄ K
+
-ATPase to hypertrophy and its
marker genes in cardiac myocytes. J Biol Chem 274,
19323–19328.
37 Elimadi A, Bouillot L, Sapena R, Tillement JP &
Morin D (1998) Dose-related inversion of cinnarizine
and flunarizine effects on mitochondrial permeability
transition. Eur J Pharmacol 348, 115–121.
MccJ25 induces the opening of mitochondrial transition pore M. Niklison Chirou et al.
4096 FEBS Journal 275 (2008) 4088–4096 ª 2008 The Authors Journal compilation ª 2008 FEBS