From the Biomolecular Science Center and Department
of Molecular Biology and Microbiology, University of Central Florida,
Orlando, Florida 32826, § Morphochem AG Gmunder
Strasse 37-37a, Munich 81379, Germany, ¶ Center for Apoptosis
Research, Kimmel Cancer Institute, Thomas Jefferson University,
Philadelphia, Pennsylvania 19107, and
Renal Division, Brigham
and Women's Hospital, Boston, Massachusetts 02115
Received for publication, December 17, 2002, and in revised form, January 14, 2003
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ABSTRACT |
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Omi/HtrA2 is a mammalian serine protease with
high homology to bacterial HtrA chaperones. Omi/HtrA2 is
localized in mitochondria and is released to the cytoplasm in response
to apoptotic stimuli. Omi/HtrA2 induces cell death in a
caspase-dependent manner by interacting with the inhibitor
of apoptosis protein as well as in a caspase-independent manner that
relies on its protease activity. We describe the identification and
characterization of a novel compound as a specific inhibitor of the
proteolytic activity of Omi/HtrA2. This compound (ucf-101) was isolated
in a high throughput screening of a combinatorial library using
bacterially made Omi-(134-458) protease and fluorescein-casein as a
generic substrate. ucf-101 showed specific activity against Omi/HtrA2
and very little activity against various other serine proteases. This
compound has a natural fluorescence that was used to monitor its
ability to enter mammalian cells. ucf-101, when tested in caspase-9
( Omi/HtrA2 is a human serine protease that has extensive
homology to bacterial high temperature requirement A
(HtrA)1 proteins (1,
2). Bacterial HtrAs have a dual function, acting as chaperones
at normal temperatures and as proteases at elevated temperatures and
removing damaged or denatured proteins, allowing the recovery and
survival of bacteria following stress (3, 4). Mammalian Omi/HtrA is a
ubiquitous protein, although tissue-specific alternatively spliced
forms have been reported (2, 5). Recent studies (6-10) described Omi
as a mitochondrial protein that upon induction of apoptosis is released
to the cytoplasm where it binds XIAP (X chromosome-linked inhibitor of
apoptosis protein) resulting in caspase-9 activation. In this regard,
Omi resembles Smac/DIABLO, which also binds IAPs and as a result
activates caspase-9 (11, 12). Omi protein is synthesized as a precursor that is processed in the mitochondria to produce the mature protein. This processing exposes an internal tetrapeptide motif, AVPS, at the
amino terminus. A similar motif is found in all IAP-binding proteins,
including the Drosophila Grim, Reaper, Hid, and
Sickle (13-16). Processing of the precursor Omi polypeptide is an
intramolecular reaction and requires an intact protease
domain.2 Omi can also induce
apoptosis in a caspase-independent pathway that relies entirely on its
ability to function as a serine protease (6, 7, 9). Neither the
mechanism nor the significance of this function of Omi is clearly
understood. In order to investigate the caspase-independent mechanism
of apoptosis initiated by Omi, we isolated specific inhibitors of its
proteolytic activity. One such family of synthetic compounds was
identified and is described in this paper. These heterocyclic compounds
showed significant and specific activity against the protease in an
in vitro assay. A compound from this family, which exhibited
the highest activity against Omi, was selected for more detailed
studies. This compound (ucf-101) naturally fluoresces and easily enters
mammalian cells, allowing its use in in vivo experiments.
Our results clearly showed ucf-101 had a profound effect on the
activity of Omi and could substantially inhibit its ability to induce
caspase-independent apoptosis in caspase-9 ( The ucf-101 inhibitor can be used as a tool to dissect the two
different activities (caspase-dependent versus
caspase-independent) of Omi and their respective contribution to
apoptosis in various biological settings. Furthermore, ucf-101, or a
similar compound, may be useful as an anti-apoptotic drug that would
specifically target caspase-independent cell death under clinical conditions.
Preparation of FITC-labeled, Dephosphorylated
Casein--
Dephosphorylated-casein (Sigma, 3 ml of 2 mg/ml stock
solution) was used in a dialysis cassette (Pierce) placed in labeling buffer (0.03 mg/ml FITC, 50 mM sodium borate, pH 9.3, 40 mM NaCl) and kept for 48 h at 4 °C in the dark with
constant stirring. After labeling, the FITC-dephosphorylated casein
(Dcasein-FITC) was further dialyzed against 50 mM Tris-HCl,
pH 7.5, and 50 mM NaCl to remove any residual unlabeled
FITC. The Dcasein-FITC conjugate was recovered from the dialysis
cassette and stored at 4 °C in the dark.
Expression and Purification of MBP-Omi and MBP-L56
Proteases--
PCR was used to amplify DNA sequences corresponding to
Omi/HtrA2 (amino acids 134-458) and L56/HtrA1 (amino acids 140-480). The PCR products were digested with MfeI and XbaI
and cloned in the corresponding EcoRI and XbaI
restriction sites of pMAL-p2X vector (New England Biolabs).
MBP-Omi-(134-458) and MBP-L56-(140-480) proteases were expressed in
TB1 Escherichia coli (New England Biolabs) and purified on
an amylose binding affinity column as described by the manufacturer
(New England Biolabs). The concentration of purified proteases was
determined using the Bradford assay.
Expression and Purification of His-tagged
Omi-(134-458)--
PCR was used to amplify the DNA sequence
corresponding to Omi/HtrA2 (Omi-(134-458)). The PCR product was cloned
in-frame in the bacterial expression vector pET-28 (Novagen). For
bacterial expression, BL21 (DE3) (Novagen) bacteria were transformed
with pET-Omi-(134-458). Single colonies were grown overnight in LB medium containing kanamycin. The overnight culture (1 ml) was used to
inoculate 1 liter of LB medium, and growth was continued at 37 °C
until the A600 was ~0.8. At this time,
1 mM isopropyl-1-thio- Proteolytic Activity of Omi Using Dcasein-FITC as
Substrate--
Opaque microtiter plates (Corning Glass) were used in
order to minimize background absorbance. A typical assay included 10 µg of Dcasein-FITC substrate, 2 µg of MBP-Omi-(134-458) in assay buffer (20 mM
Na2HPO4/NaH2PO4, pH
7.4, 200 mM NaCl, 5% glycerol) in a final volume of 100 µl. Dcasein-FITC conjugate solution (50 µl of a 0.2 µg/µl stock
solution) was added in each well using a Multidrop 384 multiple
dispenser (Labsystems) and incubated in the Wallac 1420 Victor2 Multilabel Counter at 37 °C for 15 min. After
this time, 50 µl of MBP-Omi-(134-458) protease (40 ng/µl) was
added to each well. The fluorescence (535 nm) of the reactions was
recorded every 5 min for a 30-min period.
Combinatorial Library Screening--
A Pharma Library Collection
(Nanosyn) was screened for compounds, which may inhibit the proteolytic
activity of MBP-Omi-(134-458) in an in vitro
assay. Each compound, 10 µM final
concentration, was preincubated with 50 µl of MBP-Omi-(134-458) (2 µg) for 15 min at 37 °C. After this time, Dcasein-FITC solution
(10 µg) was added, and the change in fluorescence was read every 5 min during a 30-min period using a 535-nm bandpass filter. The
proteolytic activity of MBP-Omi-(134-458) was expressed as the
percentage inhibition, where 100% refers to the activity in the
absence of inhibitor (Me2SO replaced the inhibitor
in the assay).
Activity of Several ucf Analog Compounds Against
MBP-Omi-(134-458)--
The assay was performed as described above
using Dcasein-FITC as substrate. 2 µg of MBP-Omi-(134-458) was
incubated with various concentrations of ucf-101, ucf-102, ucf-103, or
ucf-104 for 15 min. After this time 10 µg of Dcasein-FITC substrate
was added, and the reaction proceeded for an additional 30 min. The
activity of MBP-Omi-(134-458) was monitored at 535 nm.
Determination of IC50 for ucf-101 and
ucf-102--
The IC50 value for each of the two selected
inhibitors was obtained using His-Omi-(134-458) and the EnzCheck assay
kit (Molecular Probes) that contains BODIPY FL-casein as a generic
substrate. Briefly, 35 µl of His-Omi-(134-458) (500 nM)
diluted in 20 mM Na2HPO4/NaH2PO4, pH
7.4, 200 mM NaCl, 5% glycerol was incubated with 5 µl of
various concentrations of the inhibitors (0.1-1000 µM)
in 100% Me2SO (final concentration of Me2SO
was 10%); 10 µl of BODIPY-FL casein (2.5 µM final
concentration) diluted in buffer was added. The assay was carried out
using 384-well microtiter plates. Fluorescence was monitored
continuously for 10 min at 37 °C on a Tecan SpectraFluorPlus
microtiter plate reader (Tecan, Crailsheim, Germany) at an excitation
wavelength 485 nm/emission wavelength 530 nm. IC50 values
were calculated using the GraFit 4 program (Erithacus Software,
Middlesex, UK).
Activity of ucf-101 against Various Serine Proteases--
The
amount of the inhibitor giving a 50% (IC50) decrease of
the enzyme activity compared with the control reaction was estimated for various serine proteases. Briefly, 35 µl of the indicated enzyme
diluted in the corresponding buffer was incubated for 10 min with 5 µl of various concentrations of inhibitor (0.1-1000 µM) in 50% Me2SO, 50% buffer. After this
time, 10 µl of the corresponding peptidic colorimetric or fluorogenic
substrate diluted in buffer was added. In the case of the FVIIa/TF
assay, a preincubation of both proteins for 10 min at room temperature
was performed prior to addition of the inhibitor to allow complex
formation. For this experiment the following materials were used:
canine FXa, rat FXa, and rabbit FXa (Enzyme Research Laboratories);
trypsin from bovine pancreas and human thrombin (Sigma); human plasmin, human t-PA, human u-PA, and human kallikrein (Calbiochem-Novabiochem); human APC and recombinant human factor VIIa (American Diagnostica); and
recombinant tissue factor (Dade Behring). The substrates used are as
follows: spectrozyme fXa fluorogenic (American Diagnostica); kallikrein
fluorogenic substrate; protein C-activated substrate (Calbiochem);
Chromozym X, Chromozym PL, and Chromozym t-PA (Roche Diagnostics);
I-1045, I-1140, and I-1140 (Bachem).
The amount of each enzyme and its corresponding substrate
used in the assay was canine FXa (2 nM), spectrozyme fXa
fluorogenic (20 µM), rat FXa (5 nM),
spectrozyme fXa fluorogenic (25 µM), rabbit FXa (5 nM), spectrozyme fXa fluorogenic (15 µM),
kallikrein (5 nM), kallikrein fluorogenic substrate (50 µM), trypsin (2.5 nM), Chromozym X (150 µM), plasmin (5 nM), I-1045 (100 µM), thrombin (4 nM), Chromozym PL (150 µM), FVIIa/tissue factor (15 nM/3
nM), Chromozym t-PA (500 µM), t-PA (100 nM), I-1140 (200 µM), u-PA (3 nM), I-1140 (150 µM), activated
protein C (5 nM), and protein C-activated substrate (100 µM) in a final concentration of 5% Me2SO.
All enzymatic assays were carried out at room temperature in 384-well
microtiter plates (Nunc). Color development due to the release of
p-nitroanilide from the chromogenic substrates was monitored
continuously for 20 min at 405 nm on a Tecan SpectraFluorPlus microtiter plate reader (Tecan). Fluorescence from the release of the
coumarin derivative, aminomethylcoumarin, was measured at excitation
360 nm/emission 465 nm on the same reader. The IC50 values
were calculated using the GraFit 4 program (Erithacus Software).
Subcellular Localization of ucf-101--
HeLa cells were grown
on coverslips using F-12 (Ham's) nutrient mixture (Invitrogen)
supplemented with 10% fetal calf serum (Sigma), 2 mM
L-glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin (Invitrogen). Different concentrations of ucf-101 were
added to the cell media, and after 2 h cells were washed three
times with phosphate-buffered saline and fixed with 4%
paraformaldehyde. The coverslips were then placed on glass slides using
Fluoromount-G solution (Southern Biochemical Association). The
subcellular localization of ucf-101 was monitored using a LSM510
confocal laser-scanning microscope (Zeiss).
ucf-101 Inhibits Omi-induced Caspase-independent Apoptosis in
Caspase-9 ( Activity of Omi Protease against Dcasein-FITC--
Because no
physiological substrates for Omi protease are known, Combinatorial Library Screening--
By using an in
vitro high throughput assay system, we screened a combinatorial
library of synthetic compounds (Nanosyn). This collection represented
compounds that had commonly accepted pharmaceutical hit structures with
possible pharmacological properties. The assay was performed in
multiple 96-well plates. The inhibition activity of each compound was
expressed as the percentage of relative fluorescence change (decrease)
compared with the control (no compound). The final concentration of
each compound tested was 10 µM. Fifty two compounds
showed more than a 20% inhibition of MBP-Omi-(134-458) activity in
the initial screening, but only one compound (ucf-101) showed greater
than a 50% inhibition at the concentration tested after two rounds of
screening and selection (Table I).
Inhibition of the Proteolytic Activity of MBP-Omi-(134-458) Using
ucf-101 and Various Analogs--
We obtained several analogs of
ucf-101, and the chemical structures are shown in Fig.
1. The activity of these ucf analogs was
tested using MBP-Omi-(134-458) in the same in vitro assay system. All three analogs inhibited the proteolytic activity of MBP-Omi-(134-458) but to a lesser extent than ucf-101 (Fig.
2). ucf-104 (20 µM)
inhibited 58% MBP-Omi-(134-458) activity, whereas the same
concentration of ucf-101 inhibited more than 78% MBP-Omi-(134-458) activity. When MBP-Omi-(134-458) was preincubated with various concentrations of ucf-101, -102, -103, and -104 at 37 °C for longer periods (40 min or 1 h) inhibition was reduced, indicating the inhibitors did not irreversibly bind the enzyme (results not shown). MBP-Omi-(134-458) was also incubated with various amounts of
Dcasein-FITC in the presence of 10 µM ucf-101. The rate
of the reaction decreased by 50% when the concentration of the
substrate was doubled and even reached the rate of the control reaction
at higher concentrations of Dcasein-FITC (results not shown). These
results suggest ucf-101 is a competitive inhibitor of Omi protease.
Assay of His-Omi-(134-458) Activity Using Unlabeled Comparison of the Proteolytic Activities of His-Omi-(134-458) and
His-L56-(156-480) Proteins--
Omi/HtrA2 and L56/HtrA1 are members
of the same family of mammalian serine proteases with extensive
homology in their catalytic domains (1). We used bacterially made
His-Omi-(134-458) and His-L56-(140-480) to investigate whether
ucf-101 was able to inhibit the activity of His-L56-(140-480) in a
similar manner. Fig. 4 shows ucf-101 is
an effective inhibitor of the activity of His-L56-(140-480) but to a
lesser extent than His-Omi- (134-458).
IC50 of ucf-101 against Various Serine
Proteases--
The enzymatic activity of His-Omi and the determination
of the IC50 for ucf-101 and ucf-102 were performed as
described under "Experimental Procedures." These values are shown
in Table II. ucf-101 had an
IC50 of 9.5 µM, and ucf-102 had an
IC50 of 45.9 µM.
The specificity of ucf-101 was determined using several unrelated
serine proteases. Their susceptibility to inhibition by ucf-101 was
tested. The IC50 values for ucf-101 inhibitor from these
experiments are shown in Table III. These
results suggest ucf-101 has very high specificity for the Omi
protease.
Intracellular Localization of ucf-101--
To investigate the
potential use of ucf-101 inhibitor in in vivo experiments,
we tested its ability to enter mammalian cells. ucf-101 has natural
fluorescence at 543 nm that was used to detect the presence of the
compound. HeLa cells were treated with different concentrations of
ucf-101 and observed by confocal microscopy. Intense red fluorescence,
due to the presence of ucf-101, was observed in the cytoplasm of the
treated cells (Fig. 5)
ucf-101 Can Inhibit Omi-induced Caspase-independent
Apoptosis--
Mouse embryo caspase-9 ( Omi/HtrA2 is a mitochondrial serine protease that is released to
the cytoplasm upon induction of apoptosis. In the cytoplasm, Omi binds
to XIAP and relieves its inhibition of caspase-9 (6, 7, 9). In this
respect, Omi acts in a manner similar to Smac/DIABLO, another
mitochondrial protein that also binds to XIAP (11, 12). This
interaction with XIAP is mediated via an AVPS motif that is exposed at
the amino terminus of mature Omi protein after processing (6, 7). Omi
is also able to induce apoptosis in a caspase-independent manner that
exclusively relies on its ability to function as a protease (6-10).
This caspase-independent pathway is not well understood; its
contribution to overall cell death is not known, and the role of Omi as
a protease in this pathway is not clear. Furthermore, although the
catalytic domain of all HtrAs including Omi protein is conserved from
E. coli to humans, the AVPS tetrapeptide is replaced at
least in bovine Omi by SVLG (18). This suggests the IAP binding
activity of human HtrA2 that leads to caspase-dependent
apoptosis may not be critical to the overall function of Omi. Because
the caspase-independent pathway relies on the ability of Omi to
function as a protease, it suggests that proteolytic cleavage of
specific proteins is involved. This cleavage might inactivate and
remove apoptotic inhibitors, or it may activate precursor proteins
whose function might be necessary for caspase-independent cell death.
In order to investigate the contribution of the proteolytic activity of Omi to its overall pro-apoptotic function, we decided to screen for
specific inhibitor(s) of its activity. One such specific inhibitor was
identified and called ucf-101. This heterocyclic compound was able to
inhibit the proteolytic activity of Omi in vitro in a very
specific and reversible manner. The specificity of ucf-101 was tested
against a panel of several unrelated serine proteases, and no
significant activity was detected. When L56/HtrA1 was used in the same
assay, ucf-101 showed specific inhibition against this protease.
L56/HtrA1 belongs to the same family of proteases as Omi, and they both
share extensive homology throughout their respective catalytic domains
(1). The normal function of L56/HtrA2 is not known, and unlike Omi that
localizes to mitochondria, L56 is secreted (19). The natural
fluorescence of ucf-101 was used to monitor its ability to enter
mammalian cells. This property is essential for ucf-101 to be useful
for in vivo experiments designed to test inhibition of Omi
that is intracellular. To test the ability of ucf-101 to inhibit the
proteolytic activity of Omi in vivo, we used an assay where
transient overexpression of a cytoplasmic form of Omi
induces caspase-independent cell death (7). The cells used in this
assay were caspase-9 (/
) null fibroblasts, was found to inhibit Omi/HtrA2-induced cell death.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
) null fibroblasts
(17).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-galactopyranoside was added, and the culture was placed in a shaking incubator overnight at 20 °C. Bacteria were harvested by centrifugation and lysed in a
buffer containing 20 mM Tris-HCl, pH 7.5, 500 mM NaCl, 100 µg/ml lysozyme, and a protease inhibitor
mixture (Sigma). The bacterial suspension was then sonicated, and the
soluble fraction was purified using nickel-nitrilotriacetic acid
His-Bind resin (Novagen). The purity of the His-Omi-(134-458) protein
was monitored by SDS-PAGE and Coomassie Blue staining of the resulting gel.
/
) Null Fibroblasts--
Caspase-9 (
/
)
cells were transfected with either pEGFP-N1 vector
(Clontech) or M-Omi-GFP that encodes a cytoplasmic
form of Omi/HtrA2 (7). Transfected cells were kept in medium containing different concentrations of ucf-101. This medium, including the inhibitor, was replaced every 12 h. After 36 h, cells were
stained with propidium iodide and 4',6-diamidino-2-phenylindole as
described (7). Normal and apoptotic GFP-expressing cells were counted using fluorescence microscopy.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-casein was
used as a generic substrate in in vitro assays to monitor
the protease activity of Omi. For high throughput screening, we
developed a new assay that uses a mixture of
-casein,
-casein, and dephosphorylated casein (Dcasein) coupled to FITC, as a substrate. The use of Dcasein-FITC increases the fluorescence during the reaction
with less fluctuation and lower background (data not shown). A typical
assay uses 10 µg of Dcasein-FITC and 2 µg of MBP-Omi-(134-458) in
100 µl of reaction buffer (20 mM sodium phosphate buffer,
pH 7.5, 200 mM NaCl, and 5% glycerol) at 37 °C for 30 min. There was a rapid increase in fluorescence for the first 20 min
followed by a slower increase for the remaining 10 min of the assay.
Various concentrations of substrate, Dcasein-FITC, as well as
MBP-Omi-(134-458) were used for kinetic studies.
Screening for inhibitors of the proteolytic activity of Omi
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Fig. 1.
Chemical structures of ucf-101, ucf-102,
ucf-103, and ucf-104.
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Fig. 2.
Inhibition of MBP-Omi-(134-458) by ucf-101
and several other ucf analogs. The assay was performed with 10 µg of Dcasein-FITC incubated with 2 µg of MBP-Omi-(134-458) in the
presence of various concentrations of ucf-101 and three analogs,
ucf-102, ucf-103, and ucf-104. , ucf-101;
, ucf-104;
,
ucf-103;
, ucf-102.
-Casein and
SDS-PAGE Analysis--
The inhibitory effect of ucf-101 on the
activity of His-Omi-(134-458) was monitored by incubating inhibitor
and enzyme together for 10 min at room temperature prior to the
addition of
-casein as a generic substrate. Fig.
3 shows that bacterially made
His-Omi-(134-458) had substantial activity against
-casein, and
after 30 min total degradation of the substrate occurred. ucf-101
inhibited His-Omi-(134-458) activity in a
concentration-dependent manner (lanes 4-9) when assayed for 30 min with 200 ng of His-Omi-(134-458) and 5 µg of
-casein. ucf-101 (80 µM) was able to inhibit
completely the activity of 200 ng of His-Omi-(134-458) (lane
5).
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Fig. 3.
In vitro assay of the
proteolytic activity of His-Omi-(134-458) in the presence of different
concentrations of ucf-101. The assay was performed at 37 °C,
and after the indicated time points the reactions were resolved on
SDS-PAGE and the gel stained with Coomassie Blue. Omi (200 ng) was
incubated with 5 µg of -casein substrate in a 20-µl reaction
volume for 30 min at 37 °C. The ucf-101 compound was preincubated
with His-Omi-(134-458) (His-Omi) protease for 10 min at room
temperature prior to the addition of
-casein substrate. Lane
1,
-casein control; lane 2, His-Omi +
-casein,
digested for 15 min; lane 3, His-Omi +
-casein, digested
for 30 min; lane 4, His-Omi + 100 µM ucf-101 +
-casein; lane 5, His-Omi + 80 µM ucf-101 +
-casein; lane 6, His-Omi + 50 µM ucf-101 +
-casein; lane 7, His-Omi + 30 µM ucf-101 +
-casein; lane 8, His-Omi + 20 µM ucf-101 +
-casein; lane 9, His-Omi + 10 µM ucf-101 +
-casein; and lane 10, prestained molecular weight
marker.
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Fig. 4.
Inhibition of the proteolytic activity of
MBP-Omi-(134-458) and MBP-L56-(140-480) by ucf-101. The assay
was performed with 10 µg of FITC-Dcasein and 2 µg of
MBP-Omi-(134-458) (MBP-Omi) or MBP-L56-(140-480) (MBP-L56) in the
presence of various concentrations of ucf-101. The increase in
fluorescence for the initial 20 min was used for comparing the
activities of MBP-Omi-(134-458) or MBP-L56-(140-480). , MBP-Omi;
, MBP-L56.
IC50 of ucf-101 and ucf-102 inhibitors on His-Omi
IC50 values of ucf-101 on various proteases
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Fig. 5.
Cytoplasmic localization of
ucf-101. Subconfluent HeLa cells were treated with 50 (A), 40 (B), and 20 µM
(C) ucf-101 and were observed using a confocal microscope.
A-C show cytoplasmic red staining due to the presence of
ucf-101, indicating cells are permeable to this chemical. D,
no staining was seen when Me2SO alone was
used.
/
) null fibroblasts
(17) were transiently transfected with pEGFP-N1 (control) or M-Omi-GFP
(7). Cytoplasmic expression of M-Omi-GFP induced apoptosis in ~70% of the transfected cells. When cells were treated with increasing concentrations of ucf-101, it was found that increasing the
concentration of inhibitor up to 10 µM gradually reduced
Omi-induced apoptosis up to 40%. ucf-101 had no effect on the control
(vector alone) transfected cells. When the concentration of the ucf-101
increased to 25 µM, the anti-apoptotic activity of the
inhibitor was compromised due to a cytotoxic side effect that caused
apoptosis both in the M-Omi-GFP, as well as control transfected cells
(Fig. 6).
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Fig. 6.
Inhibition of Omi-induced caspase independent
cell death by ucf-101. Caspase-9 ( /
) fibroblasts were
transfected with pEGFP or M-Omi-EGFP (7) and kept in media containing
different concentrations of ucf-101. The percentage of apoptotic cells
in the transfected population was determined after 36 h as
described (7).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
) null mouse embryonic fibroblasts
(17) in which overexpression of a cytoplasmic form of Omi can induce
the caspase-independent pathway of apoptosis. This form of cell death
caused by Omi relies entirely on its ability to function as a protease.
Therefore, if ucf-101 blocks the activity of Omi, it will interfere
with apoptosis of the caspase-9 (
/
) mouse embryonic
fibroblasts. When these fibroblasts were transfected with the vector
encoding the cytoplasmic Omi and treated with ucf-101, apoptosis was
dramatically reduced. ucf-101, like many known protease inhibitors, was
cytotoxic to mammalian cells when used at high concentrations.
Therefore, the ability of ucf-101 to inhibit caspase-independent
apoptosis at low doses was compromised by its cytotoxicity at higher
doses. This toxicity was seen only at high concentrations of the
chemical and does not exclude its use in in vivo experiments
where it exhibits activity against Omi at much lower concentrations.
ucf-101 will be a useful tool to elucidate the role of Omi in
caspase-independent cell death. Several clinical conditions, such as
neurodegenerative diseases, show excessive caspase-independent cell
death (20, 21). If Omi through its proteolytic activity plays a role in the pathogenesis of any of these clinical conditions, ucf-101 or a
similar compound would be useful for therapeutic intervention against
untimely or excessive cell death.
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ACKNOWLEDGEMENTS |
---|
We are grateful to the members of the Zervos lab and Patrice Connel for comments, suggestions, and critical reading of the manuscript. The excellent technical assistance of Petra Baums and Claudia Burkhart is appreciated. We also thank R. Crowl for the L56/HtrA1 cDNA.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants R01 DK55734-01 (to A. S. Z.) and R01 AG13487 (to E. S. A.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
** To whom correspondence should be addressed: Biomolecular Science Center, Dept. of Molecular Biology and Microbiology, University of Central Florida, 12722 Research Pkwy., Orlando, FL 32826. Tel.: 407-737-2583; Fax: 407-384-2816; E-mail: azervos@mail.ucf.edu.
Published, JBC Papers in Press, January 15, 2003, DOI 10.1074/jbc.M212819200
2 L. Cilenti, A. Yerkes, L. Musumeci, and A. Zervos, submitted for publication.
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ABBREVIATIONS |
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The abbreviations used are: HtrA, high temperature requirement A; IAP, inhibitor of apoptosis protein; XIAP, X chromosome-linked inhibitor of apoptosis protein; t-PA, tissue plasminogen activator; urokinase-type plasminogen activator, FITC, fluorescein isothiocyanate; MBP, maltose-binding protein; GFP, green fluorescent protein.
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