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INTRODUCTION |
In mammals, programmed cell death can be initiated by three
distinct pathways: (i) the extrinsic pathway, which can be triggered by
ligation of death receptors and subsequent caspase 8 activation; (ii)
the intrinsic pathway, which is initiated by cellular stress followed
by activation of caspase 9; or (iii) the granzyme B pathway, where the
cytotoxic cell protease granzyme B is delivered to sensitive target
cells. Each of these pathways converges to a common execution phase of
apoptosis that requires the activation of caspases 3 and 7 from their
inactive zymogen form to their processed, active form (1, 2). The
apical activators, caspase 8 and 9, and granzyme B all have a primary
specificity for cleavage at Asp297 (caspase 1 numbering convention), located in a region that delineates the large
and small subunits of active caspases 3 and 7.
The activation of the cell death pathway depends on both the triggering
stimulus and the cell type (3), and in many forms of apoptosis
cytochrome c release from mitochondria is important for
activation of downstream caspases (4). The Bcl-2 protein family
contains both pro- and anti-apoptotic members that can act as an
upstream checkpoint of caspase activation at the level of the
mitochondria by controlling cytochrome c release. Bid, a
pro-apoptotic member of the family, has recently been identified as a
target for proteolytic cleavage by caspase 8 and granzyme B (5-8).
Activated caspase 8 cleaves Bid at Asp59 to trigger
translocation from the cytosol to the mitochondria where it promotes
cytochrome c release.
Direct cleavage of both Bid and the downstream caspases can promote
death pathways; however, it is unclear to what degree specificity of
cleavage is required. For example, whereas processing of the caspase 3 and 7 zymogens at Asp297 is considered to be the dominant
physiologic pathway for activation, cleavage of pro-caspase 7 at
Gln295 is sufficient to activate the zymogen in
vitro (9). Such results suggest that alternative proteolytic
events may be sufficient to activate pro-caspases and perhaps Bid
cleavage, especially in pathologic instances where proteolysis tends to
be unregulated.
The lysosome is the primary reservoir of nonspecific proteases in the
mammalian cell. In certain pathological situations, as well as during
normal aging (10, 11), lysosomal integrity may be compromised, causing
leakage of lysosomal proteases into the cytosol. Thus, certain diseases
related to lysosomal pathology may have a primarily apoptotic
component. Several lines of evidence support this possibility: (i)
leakage of lysosomal proteases into the cytosolic compartment may be
involved in the activation of caspases (12); (ii) in Jurkat T-cells,
-tocopheryl succinate triggers apoptosis and caspase 3 activation
accompanied by lysosomal destabilization (13); and (iii) the lysosomal
protease cathepsin B has been implicated in the activation of the
proinflammatory caspases 11 (14) and 1 (15) as well as in the induction
of the nuclear morphology associated with apoptotic cells (15).
The role of lysosomal proteases in the activation of the
apoptotic pathway is unclear. To examine the possibility that
they may be involved in programmed cell death, the activity of both recombinant cathepsins and lysosomal extracts on recombinant caspases and cytosolic extracts was examined. We tested two hypotheses: that
lysosomal proteases may directly activate executioner caspases and that
lysosomal protease cleavage of Bid may separately trigger the intrinsic
apoptosis pathway.
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EXPERIMENTAL PROCEDURES |
Materials--
Phosphate-buffered saline, fetal bovine serum,
cytochrome c, Percoll, EGTA,
p-iodonitrotetrazolium violet, penicillin, streptomicin, anti-mouse and anti-rabbit horseradish peroxidase antibodies, and
4-methylumbelliferyl-2-acetamido-2-dexy-
-D-glucopyranoside were purchased from Sigma. All other chemicals were of analytical grade. Human cathepsins B (16), H (17), K, (18), L (19) S (20), and X
(21), cruzipain (22), and human granzyme B (23) were purified as cited.
Human stefins A and B (24) and human cystatins C, D, E/M, and F were
purified as described previously (25-28), and human low molecular
weight kininogen was purified as described (29). Active caspases 3, 6, 7, 8, and 10 and zymogens of caspases 3 and 7 were expressed and
purified from Escherichia coli as described (30).
Recombinant mouse Bid was purified as described previously, except that
all detergents were omitted from the preparation procedure (31). Rabbit
antisera against human caspases 3, 6, 7, 8, 9, and 10 were prepared as
described (32-34). Anti-human caspase 2 and monoclonal cytochrome
c antibody were purchased from Santa Cruz Biotechnology and
Pharmingen, respectively. The chromogenic caspase substrate
acetyl-Asp-Glu-Val-Asp-7-p-nitroanilide (Ac-DEVD-pNA)1 was purchased
from BIOMOL, while the fluorogenic substrates
benzyloxycarbonyl-Phe-Arg-7-amino-4-methylcoumarin (Z-FR-AMC),
acetyl-Asp-Glu-Val-Asp-7-amino-4-trifluorometylcoumarin (Ac-DEVD-AFC)
were obtained from Bachem and Enzyme Systems Products, respectively.
The inhibitors E-64 and
benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-fmk) were from
Peptide Research Institute and Enzyme Systems Products, respectively.
Stock solutions of the substrates and inhibitors were prepared in
dimethyl sulfoxide and stored at
20 °C for up to 12 months.
Biochemical Markers for Lysosomes and Mitochondria--
Because
the purity of lysosomes and mitochondria was paramount in this study,
we analyzed all purifications with one mitochondrial and two lysosomal
markers to minimize any cross-contamination between these organelles.
For lysosomes we followed the activity of lysosomal proteases on
Z-FR-AMC substrate (35) and
-hexoaminidase using a method modified
from (36). For mitochondria, we used succinic
p-iodonitrotetrazolium reductase (37). Test samples during the purifications were treated with Triton X-100 (final concentration, 1% w/v) and spun at 9,800 × g for 5 min, and the supernatant was assayed. The enzyme assays were carried
out at 37 °C. Controls in the absence of the sample were run under
the same conditions.
Lysosomes--
A 5-µl sample of each fraction was added to a
96-well plate. The reaction started by addition of 95 µl of prewarmed
10 µM Z-FR-AMC in 100 mM phosphate buffer, pH
6.0, containing 1 mM DTT and 250 mM sucrose.
The released product was measured continuously during 5 min at 37 °C
using a fmax fluorescence microplate reader (Molecular Devices), at excitation and emission wavelengths of 355 and
460 nm, respectively. For
-hexoaminidase assays, a 25-µl sample
was added to a 96-well plate. The reaction was started by addition of
75 µl of 2 mM
4-methylumbelliferyl-2-acetamido-2-deoxy-
-D-glucopyranoside in 400 mM acetate buffer, pH 4.4, containing 250 mM sucrose. The released product was measured continuously
during 20 min at 37 °C using the fmax
fluorescence microplate reader, at excitation and emission wavelengths
of 355 and 460 nm, respectively.
Mitochondria--
A 25-µl sample of succinic
p-iodonitrotetrazolium reductase was incubated with
75 µl of 2 mM p-iodonitrotetrazolium violet in
55 mM potassium dihydrogen phosphate and 55 mM
succinic acid, pH 6.0, containing 250 mM sucrose until a
pink color caused by product formation developed. The reaction was
stopped by adding 125 µl of 10% trichloroacetic acid, and the pellet
was resuspended in 100 µl of ethanol. The sample was clarified at
16,000 × g for 5 min, and the absorbance of the
supernatant was read at 495 nm on a SpectraMAX 340 spectrophotometric plate reader (Molecular Devices).
Isolation of Mouse Lysosomes--
Lysosomes were purified from
mouse liver as described previously with several modifications (36,
38). All steps were carried out at 4 °C unless otherwise noted.
Briefly, several livers were washed with sucrose/Pipes buffer (250 mM sucrose, 20 mM Pipes, pH 7.2), resuspended
in 10 volumes sucrose/Pipes buffer and homogenized by two brief pulses
from a Brinkman Polytron homogenizer. The homogenate was centrifuged
for 10 min at 540 × g to remove nuclei and
particulates. CaCl2 (final concentration, 1 mM)
was added to the supernatant, followed by incubation for 5 min at
37 °C to disrupt the mitochondria. The supernatant was centrifuged
for 10 min at 18,000 × g, and the heavy membrane
pellet was retained. At this point, the integrity of the lysosomes was
verified by comparing the activity of the supernatant and the pellet,
with the lysosomal protease substrate Z-FR-AMC in the presence and absence of a lysosomotropic detergent (Triton X-100; final
concentration, 1% w/v). If the lysosomes were judged at least 80%
intact, the heavy membrane fraction was resuspended in sucrose/Pipes
buffer, centrifuged again for 10 min at 18,000 × g,
and resuspended in Percoll (40% w/v) in sucrose/Pipes. The Percoll
solution was centrifuged for 30 min at 44,000 × g to
form a gradient, and 1-ml fractions were collected from the bottom of
the tube and assayed for mitochondrial contamination using the
lysosomal and mitochondrial enzyme markers as described above. The
lysosomal fractions were pooled, diluted in sucrose/Pipes (1:10 v/v) to
decrease the Percoll, and pelleted by centrifugation at 17,000 × g for 10 min. The lysosomal pellet was washed, resuspended
in an equal volume of sucrose/Pipes, and stored at
70 °C.
Soluble lysosomal constituents were released by three freeze-thaw
cycles with a 15-s vortex between each cycle. The suspension was
centrifuged at 10,000 × g for 10 min to pellet the
lysosomal membranes, and the supernatant was saved.
Isolation of Mitochondria--
Mitochondria were isolated from
rat heart according to the procedure described in Ref. 39. Protein
content was determined by Bradford assay (Bio-Rad), and the
mitochondria were stored on ice at 3 mg/ml mitochondrial protein. The
mitochondria were used within 2 h of preparation.
Cell-free Extract Preparation and Detection of Endogenous Caspase
Levels--
A cytosolic extract from the human neuronal cell line
NT2/D1 was prepared as described previously (40). Protein concentration was determined using the Bradford assay (Bio-Rad), and the extract was
diluted to 10 mg/ml by the addition of potential caspase activators (cytochrome c or proteases). Samples were resolved by
SDS-PAGE on 8-18% gradient acrylamide gels, electrophoretically
transferred to Immobilon-P membrane (Millipore), and probed with
antibodies against human caspases. Caspase-antibody complexes were
detected with horseradish peroxidase conjugated goat anti-rabbit IgG
using ECL (Amersham Pharmacia Biotech).
Caspase Activation--
The activation of caspase zymogens,
whether recombinant or in NT2 cytosolic extracts, was followed
fluorometrically by monitoring AFC release from the substrate
Ac-DEVD-AFC. A 5-µl aliquot of NT2 cytosolic extract (14 mg/ml) or
zymogens of caspases 3 and 7 (70 nM) were incubated in
assay buffer (50 mM Hepes, 100 mM NaCl, 0.1%
(w/v) CHAPS, 10% (w/v) sucrose, and 10 mM DTT, pH 7.4) with potential proteolytic activators in the range of 1 nM
to 1 µM at 37 °C for 30 min, (total volume, 50 µl).
For analysis of potential activators in the lysosomal extract, 2-5
µl of extract was used in place of the purified proteases. Activation
was analyzed by adding 50 µl of the substrate Ac-DEVD-AFC (200 µM) in 96-well microplate format to a 37 °C
thermojacketed fluorescence microplate reader (Molecular Devices),
and caspase activity was determined at excitation and emission
wavelengths of 405 and 510 nm, respectively. The reaction was followed
continuously for 30 min. The steady-state hydrolysis rates were
obtained from the linear part of the curves.
The instantaneous rates of cruzipain-mediated activation for caspase
zymogens 3 and 7 were determined as described previously (9). Briefly,
caspase zymogen 3 (final concentration, 92.3 nM) or caspase
zymogen 7 (final concentration, 20 nM) were added to the
substrate, the reaction was started by cruzipain addition to final
concentrations of 50, 200, or 300 nM, and the time course was followed continuously for 30 min. In separate experiments to
characterize the NT2 cytosolic extracts, granzyme B (final concentration, 20 nM) or cytochrome c/dATP
(final concentration, 10 µM/1 mM) was added
to 40 µl of extract in the presence of Ac-DEVD-pNA (final
concentration, 100 µM). Although the NT2 extracts usually activated equally well in the presence or absence of dATP, we kept this
in as a standard procedure (41), although subsequent descriptions may
refer to cytochrome c alone. The release of
p-nitroanilide was continuously recorded during 30 min at
405 nm in 96-well microplate format using a SpectraMAX 340 spectrophotometric plate reader (Molecular Devices) thermojacketed at
37 °C.
Inhibition Studies--
The active concentration of each of the
purified lysosomal proteases was standardized by using E-64 (42), and
the active concentration of caspases was standardized by a similar
protocol utilizing Z-VAD-fmk (30). The endogenous anti-lysosomal
protease concentration in NT2 cytosolic extracts was determined by
titration using standardized proteases where 5 µl of each enzyme
(0.1-2 nM (final concentration) dissolved in 100 mM sodium phosphate, 2 mM DTT, pH 6.0) was
incubated with increasing concentrations of NT2 cytosolic extract
(0-100 mg/ml) for 30 min at 37 °C. The residual activity in the
presence of 10 µM of the lysosomal protease substrate
Z-FR-AMC was measured continuously in a thermojacketed fluorescence
microplate reader (Molecular Devices) using excitation and emission
wavelengths of 355 and 460 nm, respectively.
The effects of the cystatins were studied using human stefins A and B
(final concentration, 1 µM), cystatins C, D, E, and F
(final concentration, 300 nM) and low molecular weight
kininogen (2.9 µM) were incubated with caspases 3, 6, 7, and 8 (final concentration, 0.02-10 nM) for 30 min at
37 °C, and the residual activity on Ac-DEVD-AFC (final
concentration, 100 µM) was measured as above with
excitation and emission wavelengths of 405 and 510 nm, respectively.
N-terminal Sequencing--
Protein samples were resolved by
SDS-PAGE and transferred to Immobilon-P membrane. The membranes were
stained with Coomassie Blue for 2 min, destained, and washed
extensively with distilled water. The appropriate bands were excised
and sequenced on a 476A protein sequencer (Applied Biosystems).
In Vitro Assay for Cytochrome c Release--
An aliquot of rat
mitochondria equal to 10 µg of protein (or 15 µg of protein for
mouse mitochondria) incubated in the presence or absence of a 2.5-µl
aliquot of Bid at a final concentration of either 1 or 10 nM. Lysosomes at 1 mg/ml protein concentration were added
at the indicated volumes. The volume was supplemented to a final volume
of 25 µl with cMRM medium (250 mM sucrose, 10 mM Hepes-KOH, 1 mM ATP, 5 mM sodium
succinate, 0.08 mM ADP, 2 mM
K2HPO4, pH 7.5). The mixtures were incubated at
30 °C for 40 min with gentle shaking. The mitochondria were pelleted
by centrifugation for 5 min at 10,000 × g. The
resulting pellets were resuspended in 25 µl of 20 mM
Tris-HCl, pH 8.0, 100 mM NaCl with 5 µl of SDS sample
buffer and heated at 100 °C for 5 min. 5 µl of sample buffer was
added to the supernatant fractions. The samples were resolved on a 15%
Hi-Tris gel (43). The gel was transferred to nitrocellulose (Schleichler & Schuell) and probed with anti-cytochrome c
antibody. Antibody complexes were detected with a horseradish
peroxidase-conjugated goat-anti mouse IgG (Bio-Rad) using ECL (Amersham
Pharmacia Biotech). Several exposures were taken for each blot. Total
cytochrome c content is represented by mitochondrial samples
treated with 1% Triton X-100.
Isolation of Liver Mitochondria and Cytosol from Wild Type and
bid-deficient Mice--
The procedure is essentially conducted as
described previously (5). Livers of wild type and
bid-deficient mice (8) were homogenized with a Dounce
homogenizer in cMRM medium containing 25 µM EGTA, 0.1 mM phenylmethylsulfonyl fluoride, 2 mM
MgCl2, 2 mM DTT. The homogenates were
centrifuged at 1,000 × g for 10 min at 4 °C to
remove intact cells and nuclei, and the supernatants were further
centrifuged at 10,000 × g at 4 °C for 10 min to
precipitate the heavy membrane fractions (mitochondria). The
mitochondrial pellet was resuspended in the same buffer. Mitochondria
were kept on ice and used within 2 h of preparation. The
supernatants were further centrifuged at 100,000 × g
for another 60 min to obtain the cytosolic extracts used in the assay.
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RESULTS |
Characterization of Human NT2/D1 Neuronal Precursor--
The human
NT2/D1 teratocarcinoma cell line, a neuronal progenitor, was chosen for
generating cytosolic extracts because it responds well to a variety of
apoptotic stimuli (40, 44). Caspases 2, 3, 6, 7, 8, 9, and 10, the
human caspases currently considered to participate in apoptosis
signaling, are present in the cytosolic extract (Fig.
1A). We were able to determine some of the caspase concentrations in the cytosolic extract by semi-quantitative Western blot analysis, where standard recombinant caspases were compared with the endogenous NT2 amounts (for results see
Fig. 1A). These concentrations, although probably lower than the endogenous cytosolic concentration, support rapid caspase activation following addition of activators in vitro.

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Fig. 1.
Characterization of NT2/D1 cytosolic
extract. A shows Western blots of caspase zymogens and
their processed (activated) forms following 30 min of incubation at
37 °C in the presence of cytochrome c. Concentrations of
the caspases that we were able to measure in the cytosolic extract are
listed under the respective gel lanes. Zymogens are indicated by a
circle, and processed forms are indicated by a
star. Note that no processing of caspases 8 or 10 was
detected. B shows the activation of NT2 cytosolic extract
after addition of 10 µM cytochrome c
(solid line) or 20 nM granzyme B (dotted
line) followed spectrophotometrically during 30 min at 37 °C.
The lower dashed line indicates the background Ac-DEVD-ase
activity of the cytosolic extract.
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Caspase activation in extracts was followed after the addition of
cytochrome c, which resulted in processing of pro-caspases 2, 3, 6, 7, and 9 to their presumptive active forms, whereas the upstream caspases 8 and 10 were not processed under the same conditions (Fig. 1A). The lack of pro-caspase 8 or 10 processing is in
contrast to previous results with Jurkat T-cell extracts (45) but
consistent with the general conclusion that these initiators do not
participate in executioner caspase activation by the intrinsic
(mitochondrial) pathway. We offer no explanation for the processing of
pro-caspase 2, because its function in apoptotic pathways is still
debated (46, 47). Activation was also followed by observing the
increase in activity against the substrate Ac-DEVD-pNA (Fig.
1B), which predominantly reflects activation of pro-caspase
3, although other caspases will also contribute to the increase in
activity (48). This result is consistent with cytochrome
c-mediated activation of pro-caspase 9, which then amplifies
the cascade through the activation of downstream caspases 3, 6 and 7 (as reviewed in Refs. 2 and 4).
Caspase activation can also be achieved in extracts by adding the
serine protease granzyme B, again demonstrated by an increase in the
cleavage of Ac-DEVD-pNA (Fig. 1B). Both pathways showed the
same efficiency and can be considered to represent the maximal caspase
activity that could be achieved within the extract. All subsequent
comparisons of caspase activation by lysosomal proteases were therefore
compared with the physiologic activator granzyme B.
Proteolytic Activation of Pro-caspases 3 and 7--
Because
recombinant zymogens of caspases 3 and 7 are available (34), we were
able to use these to determine whether lysosomal proteases might
directly activate these executioner caspases. Pro-caspases were
incubated with 1, 10, 100, or 1000 nM of the respective
purified protease for 30 min at 37 °C, and the maximal caspase
activities generated (in all cases at 1000 nM protease) are
shown in Fig. 2. Because some lysosomal
proteases are irreversibly inactivated at neutral pH but are stable in
the slightly acidic range of around 6.0 (49), activations were also
conducted at pH 6.0, a level optimal for the lysosomal proteases, and
then raised to the normal cytosolic pH of 7.2 for assay of caspase activity. Duplicate assays were conducted with the activation period at
pH 7.2. The data shown from this point were all collected with
activation at pH 7.2, because activation at pH 6.0 produced no more
caspase activity than at pH 7.2. Both cruzipain and cathepsin B cleaved
Ac-DEVD-pNA, supporting previous observations of nonspecific reaction
of lysosomal proteases with ostensibly specific caspase reagents based
on similar sequences (50). These background values were subtracted
before the data were compared.

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Fig. 2.
Activation of recombinant caspase zymogens 3 and 7 by purified lysosomal proteinases. Activation of caspase
zymogens 3 (p-C3) and 7 (p-C7) by cathepsins B, H, K, L, S, and X and
cruzipain (cruz), measured with Ac-DEVD-AFC after 30 min of
incubation at 37 °C, are shown in the insets of
A and B, respectively. These degrees of
activation were compared with that generated by the physiologic caspase
activator granzyme B (GB, in the main panels of
A and B, respectively). All experiments were
performed in triplicate, and the standard deviations were less than
10% of the indicated values.
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Cathepsins B, H, K, L, S, and X were unable to activate caspase
zymogens 3 and 7 directly. In contrast, cruzipain (the cathepsin L
homolog from Trypanosoma cruzi) appeared to induce a
substantial activation of pro-caspase 3 and pro-caspase 7 (Fig. 2,
insets). However, when compared with the maximal activity
obtained by granzyme B treatment, the cruzipain activation of caspase
zymogens 3 and 7 was only 10 and 3%, respectively (Fig. 2, main
panels). The activation of pro-caspase 7 was too slow to be
measured, but we were able to determine the rate of activation of
pro-caspase 3 by cruzipain to be 1.7 × 103
M
1 s
1, with activation
following cleavage after Cys170 within the sequence
ELDC170GIETD175S, five residues upstream of the
canonical zymogen activation site (Asp175) within the
caspase 3 zymogen. The slow activation rate is unlikely to be
physiologically relevant, although it cannot be ruled out that the
intracellular parasite form of T. cruzi may utilize
cruzipain to directly activate caspases at some stage during its
presence in infected host cells. Clearly, within the wide array of
parameters used in this preliminary study, none of the purified human
lysosomal cysteine proteases can be construed as direct activators of
the execution caspases.
Activation of NT2 Cytosolic Extract by Lysosomal Proteases--
To
examine whether the purified lysosomal proteases could activate
pro-caspases other than 3 and 7, we utilized NT2 cytosolic extracts as
the source of caspase zymogens. Cathepsins B and X caused a slight
increase in caspase activity (Fig. 3,
inset). Cruzipain was significantly more efficient,
exhibiting about a 2.5-fold increase in Ac-DEVD-pNA cleaving activity
relative to background (Fig. 3, inset). Granzyme B induced
maximal activation in NT2 cytosolic extracts, and although cruzipain
was a better activator than the cathepsins, its activity was only 5%
that of granzyme B (Fig. 3).

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Fig. 3.
Activation of NT2 cytosolic extract by
purified lysosomal proteases. The inset shows the
activation of NT2 cytosolic extract by purified lysosomal proteinases
and cruzipain (cruz), measured with Ac-DEVD-AFC after 30 min
of incubation at 37 °C. The main panel compares the
efficiency of activation with the maximal activation rate obtained in
the presence of the granzyme B (GB). All experiments were
performed in triplicate, and the standard deviations were less than
10% of the indicated values.
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Because purified lysosomal enzymes showed only a minor activation of
caspases in the NT2 cytosolic extract, a lysosomal extract was next
used to explore whether additional proteases not included in our
purified repertoire could cause caspase activation in NT2 extracts. The
lysosomal extract possessed no detectable activity against Ac-DEVD-AFC
and induced a 2-fold activation of the NT2 extract (Fig.
4, inset). However this is
only 5% of the activation conducted by granzyme B (Fig. 3).

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Fig. 4.
Activation of NT2 cytosolic extract by the
lysosomal extract. An extract of mouse lysosomes was added to the
NT2 cytosolic extract, and caspase activation was measured with
Ac-DEVD-AFC after 30 min of incubation at 37 °C. The
inset shows the activation caused by mixing the extracts,
and the main panel compares this with maximal granzyme
B-mediated caspase activation. All experiments were performed in
triplicate, and the standard deviations were less than 10% of the
indicated values.
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Inhibition of Lysosomal Cysteine Proteases by the NT2 Cytosolic
Extract--
One could argue that the lack of substantial activation
of pro-caspases in the NT2 cytosolic extract by lysosomal proteases may
be due to endogenous inhibitors. To explore this, we titrated the NT2
extract against standardized proteases, which revealed that cathepsins
B, H, L, S, and X and cruzipain were inhibited in the range of 0.6-25
nM, whereas cathepsin K was not inhibited (Table
I). Clearly the presence of endogenous
caspase inhibitors can be ignored, because both granzyme B and
cytochrome c activated the extracts very well. Nevertheless,
one could argue that cystatins, being inhibitors of lysosomal cysteine
proteases, may also inhibit caspases and, therefore, caspase
activation. To check this we tested representatives of all three known
families of cystatins for inhibition of caspases 3, 6, 7, and 8. No
substantial inhibition was observed, even at vast excess of cystatins
(Table II), so we can effectively rule
cystatins out as caspase regulators.
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Table I
Inhibition of lysosomal cysteine proteinases by NT2 cytosolic extract
Total inhibitory capacities in NT2 extracts against each of the
purified, standardized lysosomal proteases are shown. NI, not
inhibited.
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Table II
Effect of cystatins on caspases
Residual activity (%) of caspases at 0.02-10 nM, in the
presence of 1 µM stefins A and B, 300 nM of
each of the other cystatins, or 2.9 µM of the
double-headed cystatin low molecular weight kininogen (LK) is shown.
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Cleavage of Bid by the Lysosomal Extract--
Because the caspase
pathway in cytosolic extracts was not activated directly by lysosomal
proteases, we explored whether lysosomal extracts could cleave protein
intermediates to play a role in caspase activation. An obvious place to
look for this is in the mitochondrial amplifier of the intrinsic
apoptosis pathway. This pathway encompasses the participation of
several members of the Bcl-2 family, of which Bid is an attractive
candidate because it is activated through proteolytic cleavage by
caspase 8 or granzyme B. Truncated Bid translocates to the mitochondria
where it is a potent inducer of cytochrome c release (5-8).
Recombinant mouse Bid was incubated in the presence of lysosomal
extract and the products resolved by SDS-PAGE along with Caspase
8-cleaved Bid for comparison (Fig. 5). A
~14-kDa cleavage product of Bid resulted from lysosome extract
exposure, with the N-terminal sequence SFNQGRIEPD, showing that the
lysosomal extract cleaves Bid at Arg65 (Fig.
5B).

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Fig. 5.
Effect of lysosomal extract on mouse
Bid. A indicates the processing of recombinant mouse
Bid by lysosomal extract as shown by electroblotting followed by
Coomassie Blue staining. A positive control of caspase 8-cleaved Bid is
shown for comparison. The lysosome cleaved product indicated by an
arrow was subjected to N-terminal sequencing to yield the
sequence SFNQGRIEPD. The location of the lysosome cleavage site is
shown in B, with the locations of the caspase 8 and granzyme
B sites shown for comparison.
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It was then important to confirm whether the Bid fragment resulting
from lysosomal extract exposure had activity on mitochondria. To test
this, mitochondria were isolated from rat cardiac tissue and incubated
in the presence of either lysosomal extract alone, Bid alone, Bid and
lysosomal extract, or Triton X-100, the last possibility representing
total cytochrome c content (Fig.
6A). Uncleaved Bid was able to
induce low levels of cytochrome c release at Bid
concentration exceeding 100 nM (data not shown) but no detectable release at 1 nM. In contrast, in the presence of
lysosomal extract, Bid at 1 nM induced cytochrome
c release from mitochondria. Lysosomal extract in the
absence of Bid had no effect on cytochrome c release. Thus,
lysosome-cleaved recombinant Bid induced cytochrome c
release to levels similar to that seen for Caspase 8-cleaved Bid.

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Fig. 6.
Cytochrome c release
following lysosomal cleavage of recombinant or endogenous Bid. In
A, mitochondria (3 µg of protein at 1 mg/ml concentration)
either in the presence or absence of BID (10 nM) and
varying volumes of lysosomal extracts were incubated for 40 min at
30 °C. All volumes were supplemented to 25 µl with cMRM, pH 7.5. The pellets and supernatants were collected, and the latter was
resolved by SDS-PAGE followed by Western blotting using an
anti-cytochrome c antibody. The signals were detected by
ECL, and the film was exposed for 30 s. Incubation with Triton
X-100 (0.5%; TX) represented total cytochrome c
content. The first four lanes represent control trials
demonstrating that neither uncleaved Bid nor lysosomal extract alone is
capable of inducing cytochrome c release. Incubation of
mitochondria with both Bid and increasing amounts of lysosomal extract
(right three lanes) induced release of at least 50% of
cytochrome c when compared with mitochondria lysed with
Triton X-100. B shows similar experiments conducted in
cytosolic extracts of mouse hepatocytes, either wild type (Bid +/+) or
Bid-deficient (Bid / ). In A, 30 µl of cytosol prepared
from either wild type (Bid +/+) or bid-deficient (Bid / ) mouse
hepatocytes were incubated with either 200 ng of recombinant Caspsae 8 or 1 to 6 µl of lysosomal extracts in the presence of 5 mM DTT at 37 °C for 30 min. The reaction mixture was
then admixed with 15 µg of liver mitochondria at 30 °C for 60 min.
The supernatants were then separated from the mitochondria by
centrifugation and subjected SDS-PAGE, followed by Western blotting
using an anti-cytochrome c antibody.
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Reduced Cytochrome c Releasing Activity in bid-deficient
Cytosol--
To further confirm that Bid plays an important role as
the substrate of lysosomal enzymes in cytochrome c release,
we prepared cytosolic extracts from wild type and
bid-deficient hepatocytes. When these cytosols were
incubated with the lysosomal extracts at different doses, the wild type
extracts demonstrated a significantly stronger potency than the
bid-deficient extracts to yield a cytochrome c
releasing activity (Fig. 6B). The lysosomal extracts were
able to activate this activity in a dose-dependent manner,
which was comparable with that generated by recombinant caspase 8. In
contrast, a modest level of cytochrome c releasing activity
was induced in bid-deficient cytosols only when severalfold
more lysosomal extracts were used. These results indicated that
although Bid might not be the only substrate of lysosomal enzymes that
could be activated to induce cytochrome c, it was certainly
a major one.
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DISCUSSION |
The concept of lysosomal constituents participating in cellular
pathology and degeneration was originally proposed by De Duve (52).
Since that time, the critical role of apoptosis in executing cell death
under normal and pathologic conditions has become clear. Multiple
studies have attempted to address the role of lysosomes in triggering
apoptosis. In speaking of lysosomes and cell death, most investigators
do not distinguish between primary lysosomes (the temporal store of
transient digestive enzymes) and endosomes (the location of protein
degradation). Our in vitro system also does not distinguish
between the two organelle types, and they are probably a mixture of
each. Both organelles contain some specific and many rather nonspecific
proteases that participate in general protein digestion and more
specific digestion for loading onto major histocompatability
complex class II for antigen presentation.
Evidence for the participation of lysosomes in cell death comes from
several sources. Oxidative stress, for example, is generally associated
with mitochondrial permeability transition (4), yet it is also readily
associated with lysosomal rupture. Thus, cell death following oxidative
stress caused by naphthazarin (53) and hydrogen peroxide (54) has been
linked to lysosome rupture, as has the death of cultured microglia in
response to 6-hydroxydopamine (55), and PC12 cells in response to serum
deprivation (56). Natural insults that damage lysosomal membrane
integrity include the Alzheimer A
1-42 amyloidogenic fragment that
accumulates in lysosomal compartments (57) preceding cell death (58).
The mechanisms of death induced by lysosomes have previously been linked to direct pro-caspase activation by lysosomal proteases (12),
although there is some disagreement because others were not able to see
this direct activation (15). Interestingly, Vandenabeele's group (14,
15) found that lysosomal proteases, and notably cathepsin B, could
efficiently process pro-caspases 1 and 11 but could only weakly process
the precursors of caspases 2, 3, 6, 7, and 14. Thus, there could be a
case for direct activation of inflammatory caspases (1 and 11) but not
the apoptotic ones. Therefore, our main goal was to re-examine the
hypothesis that lysosomal proteases may lead directly to pro-caspase
activation, in search of a mechanistic explanation for
lysosome-mediated cell death.
Previous studies have mainly addressed cleavage of pro-caspases by
lysosomal proteases and not their activation. However, because we have
demonstrated that observations of cleavage alone can be misleading as a
readout of activation, at least for other proteases (59), we focused
here on direct measurements of caspase activation. We were surprised to
see that the candidate lysosomal proteases and extracts of mouse
lysosomes were only able to carry out minimal direct caspase
activation, even at very high concentrations. It is possible to see how
one may obtain the impression that lysosomal proteases activate caspase
zymogens, because slight increases in caspase activity were detected
when cathepsin B or X or lysosomal extracts were added to NT2 cytosolic
extracts (Figs. 3 and 4, insets). However, when these are
compared with the rate achieved by granzyme B, the activities pale into
insignificance. Some of the lysosomal proteases, notably cathepsins B
and L, would have been rapidly inactivated at the test pH of 7.2, but
this was taken into account by control experiments at a more permissive
pH of 6.0. Interestingly, there seems to be nothing prohibiting direct activation by members of the papain family, of which all the cathepsins in this study were members, because the homologous protease cruzipain did directly activate pro-caspases 3 and 7, albeit rather weakly. Therefore, we are left with the speculation that the endogenous lysosomal proteases have been negatively selected against for direct
caspase activation.
The triggering mechanism of death must have another explanation. It is
certainly possible that lysosomal death may sometimes be more necrotic
than apoptotic (60), yet there is plenty of evidence that definitive
morphology and biochemistry of apoptosis is activated in experimental
paradigms of lysosomal dysfunction (12, 15, 56, 61). Accordingly, we
looked elsewhere for initiators of apoptosis that would require
proteolysis for their activation. The best characterized example is Bid
(5-8), and it is clear from our studies with recombinant Bid and
endogenous material that this protein can play a role as the lysosomal
protease target. Indeed, the dampened release of cytochrome
c in extracts from hepatocytes of mice ablated for the
bid gene confirms this conjecture. Bid may not be the only
cytosolic trigger for lysosome-mediated cytochrome c
release, but it is clearly one of them.
The lysosome is a rich source of proteolytic enzymes, and one or more
of these proteases cleave and activate Bid. The activation was not
prevented by pretreating lysosomal extracts with E-64, the broad range
inhibitor of the lysosomal proteases cathepsins B, H, K, L, S, and X
(data not shown). Therefore, the activity is unlikely to be due to
these proteases or a close homolog. The cleavage site
(Arg65) within Bid was unique, at least as far as could be
deduced by Edman degradation, yet cleavage after Arg does not
necessarily mean that the protease(s) is Arg-specific. It is equally
possible that the protease(s) perceives other determinants in the
immediate vicinity. Interesting, although indirect, evidence places
lysosomal aspartic proteases, including cathepsin D in certain
apoptosis pathways (53, 62, 63), although cathepsin D itself would not
seem to have the desired substrate specificity to recognize the Bid
target sequence because it prefers to cleave between hydrophobic residues (64). One clue to the possible identity of the lysosomal protease comes from work on an inherited form of progressive myoclonus epilepsy, where the defect is a complete deficiency in stefin B (65).
The disease has been modeled by ablating the mouse stefin B gene, which
resulted in ataxia, myoclonic epilepsy and cerebellar apoptosis (66).
Thus, stefin B, which inhibits many lysosomal cysteine proteases
in vitro (51, 67), is required to avoid pathologic
cerebellar apoptosis with the likely explanation that it normally
regulates protease(s) that cause the defect. Because we show that
stefin B, or other cystatins for that matter, do not inhibit caspases
3, 6, 7, or 8, we concur that stefin B, given its in vitro
specificity, may act as an inhibitor of pro-apoptotic lysosomal
protease(s) (66).
Although Bid cleavage is the most likely route for lysosome-directed
apoptosis, we do not rule out other mechanisms. For example, cleavage
of the extended loop segment of Bcl-XL by caspases has been
demonstrated to accelerate cytochrome c release in
vitro (68), and it is possible that lysosomes may also target the same loop. Nevertheless, this would be of secondary importance given
the largely impaired cytochrome c release in Bid-deficient cytosols. It is also possible that lysosomal proteases that are not
released by the conditions used in our study may directly activate
caspases, although such entities are still hypothetical.
The mechanism that Bid uses to induce apoptosis is still unclear,
although it is likely that removal of the N-terminal helices 1 and 2 allows the protein to translocate from the cytosol to the mitochondria
where it promotes cytochrome c release (5-8). This
relocation to the mitochondrial membranes may be promoted by an
increase in hydrophobicity because loss of the N-terminal helices
results in a protein that is predicted to have an increased hydrophobic
surface area, and this form may favor membrane insertion (31, 69).
Alternatively, the cleavage also results in an increased exposure of
the BH3 domain, which may promote protein-protein interactions that
affect other proteins involved in cytochrome c release,
particularly Bak (70) and also Bax (71). Regardless of the mechanism,
proteolytic cleavage seems to be essential. Activated caspase 8 cleaves
Bid at Asp59 (6, 7), granzyme B cleaves at
Asp75 (6), and lysosomes cleave at Arg65 (Fig.
7). These sites encompass a region of the
protein that is highly flexible because it does not adopt a fixed
conformation as determined by NMR (69, 72). Such regions are excellent targets for proteolysis, because most proteases require flexible protein loops to adapt to their substrate clefts (73). We speculate that the high mobility of this region allows general proteolysis to
occur, not just the selective proteolysis of the highly specific caspase 8 and granzyme B. Thus, we propose Bid to be a general sensor
of proteolysis by endopeptidases and that the loop shown in Fig. 7 acts
as a "bait loop," allowing cells to respond to adventitious and
potentially damaging proteolysis by triggering the built-in apoptotic
suicide program. Indeed, this property would not be restricted to
lysosomal proteases but may also encompass proteases from other
organelles, or even pathogens, that inappropriately enter the cytosol
during pathologic episodes.

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Fig. 7.
Three-dimensional model of Bid and the
location of the Bid bait loop. NMR structures of Bid demonstrate
that the loop connecting helix 2 (h2) with helix 3 (h3) is extremely mobile and therefore ideal for protease
recognition. Cleavage within this loop allows hydrophobic residues in
helix 3, including the BH3 domain, to trigger the intrinsic apoptotic
pathway. It follows that any protease that could cleave in this loop,
the bait loop of Bid, should be able to initiate the apoptotic
pathway, and, therefore, Bid may be considered a general sensor of
cytoplasmic endoproteolysis.
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