(Received for publication, October 13, 1995; and in revised form, November 30, 1995)
From the
Cysteine proteases of the interleukin 1 Converting Enzyme
(ICE)/CED-3 family have been implicated in the effector process of
apoptosis in several systems, including Fas-mediated apoptosis. We have
recently isolated and partially characterized a protease present in
extracts from anti-Fas antibody treated Jurkat T cells that promotes
apoptotic changes in isolated nuclei (Schlegel, J., Peters, I., and
Orrenius, S.(1995) FEBS Lett. 364, 139-142). We now show
that this protease cleaves poly(ADP-ribose) polymerase (PARP) with high
efficiency and specificity. Both PARP proteolysis and the proapoptotic
effects of the protease are inhibited by nanomolar concentrations of a
selective inhibitor of apopain (CPP32), while an inhibitor of IL-1
converting enzyme is much less effective, requiring micromolar
concentrations for the inhibition of the isolated protease. Kinetic
analysis of the isolated protease reveals kinetic constants similar to
those reported for apopain. The isolated protease is recognized by
antibodies specific for CPP32/apopain but not by an anti-ICE antibody.
Furthermore, a selective inhibitor of apopain prevents Fas-induced
apoptosis in intact Jurkat T cells. We therefore conclude that
CPP32/apopain is activated in Fas-induced apoptosis.
Apoptosis is an important and well controlled form of cell death observed under a variety of physiological and pathological conditions. Inappropriate apoptosis may be involved in many diseases such as Alzheimer's disease, immune deficiency and autoimmune disorders, leukemias, lymphomas, and other malignancies. Therefore, the control of apoptosis is an important potential target for therapeutic intervention (1, 2, 3, 4) .
Apoptosis is
characterized by a series of distinct morphological and biochemical
changes which seem to be well conserved through evolution. One of the
best described proapoptotic genes, CED-3, encodes a Caenorhabditis
elegans protein that is highly homologous to mammalian
interleukin-1 converting enzyme (ICE), (
)the first
identified member of a new class of cysteine proteases with a near
absolute specificity for aspartic acid in the S
subsite(5, 6, 7, 8) .
Recent work has indicated the involvement of a protease belonging to the ICE/CED-3 family in Fas-mediated apoptosis(9, 10, 11) . We have previously used a reconstituted in vitro system(12, 13) containing cytosolic extracts from Jurkat T cells undergoing Fas-induced apoptosis to partially purify a protease that promotes apoptotic changes in isolated nuclei(14) . In the absence of 1,4-dithiothreitol (DTT), this proteolytic activity was inhibited by several serine protease inhibitors, iodoacetamide and VAD-FMK, while E64 was not inhibitory(14) . This inhibitor profile is consistent with the protease being a member of the ICE/CED-3 family(6, 15) . In the present work we demonstrate that this protease, which appears to play a crucial role in Fas-induced apoptosis, is not ICE itself but another member of the ICE/CED-3 family, namely CPP32/apopain.
Anti-human Fas antibody (IgM, clone CH-11) was purchased from AMS Biotechnology, Sweden. RPMI 1640 medium, fetal calf serum, glutamine, penicillin, streptomycin, and low-melting-point agarose were obtained from Life Technologies, Inc. (Paisley, Scotland, UK). All other chemicals were from local suppliers and of the highest available grade of purity.
In cytosolic extracts prepared from anti-Fas-treated Jurkat T
cells, elevation of a PARP cleavage activity is an early event first
detectable 15 min after adding an anti-Fas antibody to the cells (Fig. 1a). By comparison, fragmentation of nuclear DNA
into high molecular weight fragments starts at about 30 min (Fig. 1d), while the formation of internucleosomal DNA
fragments cannot be detected earlier than 45 min after antibody
addition (not shown). We could not detect any proteolytic activity
capable of cleaving pro-IL-1 in extracts taken at different time
points during incubation with anti-Fas antibody (Fig. 1b), even at high concentrations of the extracts
(not shown). Furthermore, these cells contained no ICE protein that
could be recognized by immunoblotting or immunoprecipitation (not
shown). This clearly argues against the involvement of ICE itself in
Fas-mediated apoptosis in Jurkat T cells. However, the early elevation
of a proteolytic activity that cleaves PARP (Fig. 1a)
implies the involvement of another enzyme of the ICE family, apopain (17) (prICE/CPP32(18, 19) ), an ICE-like
protease reported to cleave PARP(17, 18) . This
protease is synthesized as a 32-kDa precursor (p32) that is
proteolytically activated to form a mature enzyme composed of 17-kDa
(p17) and 12-kDa (p12) subunits(17) . Immunoblots using
antibodies specific for the p17 and p12 subunits of apopain show the
presence of the dormant 32-kDa precursor in untreated Jurkat cells (Fig. 1c). Following anti-Fas activation, this
precursor was rapidly broken down into active apopain (Fig. 1c) coincident with the onset of the PARP
cleavage activity. The precursor was initially processed to p12 and a
20-kDa intermediate (p20) (detectable by 30 min), followed by slower
conversion of p20 to p17. Both p20 and p17 have catalytic activity as
shown by their ability to be labeled with a biotinylated irreversible
DEVD-acyloxymethyl ketone active site probe of apopain with
biotinylation found as early as the 15-min time point (not shown). The
proapoptotic protease isolated from anti-Fas-treated Jurkat T cells was
found to contain primarily p12 and p17 (Fig. 1c),
indicating that apopain was the protease that had been purified. The
fact that the 32-kDa CPP32-precursor of apopain is converted so quickly
to the subunits that comprise active apopain, with maximal activation
of apopain occurring within 60 min after anti-Fas treatment of the
cells (Fig. 1a and Table 1), might explain why
Fas-mediated apoptosis in Jurkat T cells is that much faster than other
model systems of apoptosis.
Figure 1:
Time course of events in
anti-Fas-treated Jurkat T cells. a, elevation of PARP cleavage
activity; b, lack of pro-IL-1 cleavage activity; c, immunoblot analysis of the degradation of the CPP32
precursor into the p20, p17, and p12 subunits and of the partially
purified protease from anti-Fas-treated Jurkat T cells (Jurkat
protease) compared with purified apopain/CPP32; d, formation
of high molecular weight DNA fragments. The extraction of the cells and
the assays used were performed as described under ``Experimental
Procedures.''
When the partially purified protease
(which was unable to cleave pro-IL-1) was tested for its ability
to cleave PARP in the presence of selective inhibitors, the apopain
inhibitor Ac-DEVD-CHO (4) (K
< 1 nM) gave complete inhibition at 5 nM whereas the ICE inhibitor Ac-YVAD-CHO (6) (K
= 0.76
nM) was inactive at 5 µM and only partially
effective at 50 µM (Fig. 2a). Measurement
of the ability of the isolated protease to induce apoptotic changes in
thymocyte nuclei using a reconstituted in vitro system
revealed similar results. The ICE inhibitor Ac-YVAD-CHO was only
partially inhibitory on the protease at concentrations as high as 50
µM, but the apopain inhibitor Ac-DEVD-CHO gave at 8 nM a partial and, at 100 nM, total inhibition of nuclear
degradation (Fig. 2, b and c). In line with
these results, the cowpox virus serpin CrmA, a potent inhibitor of ICE (20) (K
< 4 pM) but not of
apopain (no inhibition at more than 1000 times higher
concentrations(17) ) did not interfere with either the PARP
cleavage activity or the nuclear degradation activity of the partially
purified protease at concentrations up to 0.1 µM (not
shown). These results provide further evidence that CPP32/apopain is
activated in Fas-mediated apoptosis.
Figure 2:
a, inhibition of the PARP cleavage
activity of the partially purified protease from anti-Fas-treated
Jurkat T cells by peptide aldehydes. [S]PARP was
incubated with purified apopain/CPP32 or the partially purified
protease in the absence or presence of Ac-YVAD-CHO, a specific
inhibitor for ICE, or Ac-DEVD-CHO, a specific inhibitor for
apopain/CPP32 as described(17) . b and c,
Ac-DEVD-CHO inhibits apoptotic changes in vitro at nanomolar
concentrations. Thymocyte nuclei were incubated with the partially
purified protease from Jurkat T cells undergoing Fas-induced apoptosis
in the absence or presence of Ac-YVAD-CHO or Ac-DEVD-CHO and DNA
degradation into high molecular weight (b) and
oligonucleosomal fragments (c) was analyzed as described under
``Experimental Procedures.''
A kinetic analysis of the extracts obtained from Fas-treated Jurkat T cells and the partially purified protease using a continuous fluorometric assay for apopain with the substrate Ac-DEVD-AMC (17) (Table 1) confirmed that the proapoptotic protease from anti-Fas-treated Jurkat T cells was apopain. The catalytic and inhibitor constants obtained for the isolated protease were virtually identical with those of purified apopain (Table 1). A quantitative analysis of the samples of the time course using a fluorometric assay indicated that significant active apopain was generated within 15 min following anti-Fas treatment (Table 1).
To test the ability of the inhibitors used in this study to block Fas-induced apoptosis in whole cells, we preincubated Jurkat T cells with the ICE inhibitor Ac-YVAD-CHO, the apopain inhibitor Ac-DEVD-CHO, and, as a further control, the corresponding peptide lacking the aldehyde group (Ac-DEVD-COOH) which does not affect apopain activity (17) . Ac-DEVD-CHO at a concentration of 120 µM was found to inhibit Fas-induced apoptosis in Jurkat T cells, whereas Ac-YVAD-CHO and Ac-DEVD-COOH were without effect (Fig. 3). Since the cells showed a viability of >95% during the entire experiment (as assessed by trypan blue exclusion; not shown), the inhibitory effect of Ac-DEVD-CHO was not due to cytotoxic effects, but rather to the inhibition of apopain. Trypan blue uptake is associated with postapoptotic necrosis and a late event in this experimental model occurring well after oligonucleosomal DNA fragmentation (>3 h) (16) .
Figure 3:
Inhibition of Fas-mediated apoptosis in
intact Jurkat T cells by the specific tetrapeptide aldehyde inhibitor
for apopain, Ac-DEVD-CHO. Jurkat T cells (10 10
/ml)
preincubated at 37 °C for 1 h with 120 µM Ac-YVAD-CHO,
120 µM Ac-DEVD-CHO, or 120 µM Ac-DEVD-COOH
before incubation with anti-Fas antibody (0.75 µg/ml) for an
additional 2 h were isolated by centrifugation, washed, and analyzed
for DNA fragmentation as described under ``Experimental
Procedures.''
In summary, our results provide strong evidence that apopain (CPP32) is activated in Fas-mediated apoptosis. Since apopain is a key enzyme initiating apoptotic events in other systems as well(17, 18) , pharmacological modulation of this protease may be of clinical importance in many diseases involving inappropriate apoptosis. Finally, we cannot exclude that additional ICE-like proteases are activated in Fas-mediated apoptosis. Although we failed to demonstrate any activation of ICE itself in anti-Fas-treated Jurkat cells, previous work has suggested that ICE may be involved in Fas-mediated apoptosis in other cell types(9, 10) . Thus, further studies are required to resolve this discrepancy.