(Received for publication, May 8, 1995; and in revised form, October 25, 1995)
From the
The Caenorhabditis elegans cell death gene, ced-3, encodes one of the two proteins required for apoptosis
in this organism. The primary sequence similarities between Ced-3 and
the mammalian interleukin-1 converting enzyme (ICE) suggest that
these two proteins may have functionally similar active sites and that
Ced-3 may function as a cysteine protease. Here we report that in
vitro transcribed and translated Ced-3 protein (p56) underwent
rapid processing to smaller fragments. Replacement of the predicted
active site cysteine of Ced-3 with serine (C364S) prevented the
generation of smaller proteolytic fragments, suggesting that the
processing might be an autocatalytic process. Peptide aldehydes with
aspartic acid at the P1 position blocked Ced-3 autocatalysis.
Furthermore, the protease inhibition profile of Ced-3 was similar to
the profile reported for ICE. These functional data demonstrate that
Ced-3 is an Asp-dependent cysteine protease with substrate specificity
similar to that of ICE. Aurintricarboxylic acid, an inhibitor of
apoptosis in mammalian cells, blocked Ced-3 autocatalytic activity,
suggesting that an aurintricarboxylic acid-sensitive Ced-3/ICE-related
protease might be involved in the apoptosis pathway(s) in mammalian
cells.
Apoptosis, or programmed cell death, plays an important role in many multicellular organisms, in morphogenesis, maturation of B and T lymphocytes, development of the nervous system, and destruction of unwanted cells, as well as in the pathogenesis of some diseases. A better understanding of this process could lead to development of specific inhibitors to treat apoptosis-related diseases. The nematode Caenorhabditis elegans provides an excellent model in which to study the genetic pathway(s) involved in apoptosis. 11 genes have been identified and implicated in apoptosis in C. elegans(1) . The functions of three of these genes are well characterized; the gene product Ced-9 prevents (2) and the Ced-3 and Ced-4 gene products promote apoptotic cell death(3, 4) .
Some of the C. elegans apoptosis genes have homologs in mammalian cells. Correlation of
the functional activities of these gene products with the activities of
their mammalian homologs could lead to a better understanding of
apoptosis in mammals. The mammalian gene bcl-2 can
functionally substitute for ced-9 in C.
elegans(5) , suggesting that the BCL-2 proto-oncogene
product is the mammalian homolog of Ced-9. Based on the amino acid
similarities between Ced-3 and the interleukin-1 converting enzyme
(ICE) (
)it was suggested that ICE might be a mammalian
homolog of Ced-3(6) . Although the ced-4 gene has been
cloned, no mammalian homolog of Ced-4 has been reported.
Ced-3 and
ICE are two members of a growing family of proteins, which also
includes the mouse product Nedd2 (neuronal precursor cell-expressed,
developmentally down-regulated gene-2)(7) , its human homolog
Ich-1 (ICE and Ced-3 homolog-1)(8) , Ich-2 (9) (also
known as TX (10) and ICE II(11) ),
CPP32/YAMA(12, 13, 14) /prICE (15) ,
ICE
III(11) , and Mch2 (mammalian Ced-3 homolog
2) (16) . Functionally, overexpression of these
Ced-3/ICE-related proteins in mammalian or insect cells induces
apoptosis. Since ICE functions as a cysteine protease with an absolute
requirement for aspartic acid at the P1
position(17, 18) , it has been proposed that these
Ced-3/ICE-related proteins function as Asp-dependent cysteine
proteases. In fact, the catalytic cysteine and histidine as well as the
residues defining the P1 carboxylate binding pocket of ICE, as
determined by the three-dimensional x-ray crystal structure, are
conserved in all these Ced-3/ICE-related
proteins(19, 20) .
ICE converts biologically
inactive 31-kDa proIL-1 to its biologically active 17-kDa form.
ICE is synthesized as a 45-kDa (p45) zymogen and is processed to
generate the p20 and p10 subunits that comprise the active
heterodimeric enzyme(17) . Generation of the active enzyme
requires four cleavages within p45 ICE (between residues 103 and 104,
119 and 120, 297 and 298, and 316 and 317). These cleavages occur
between the Asp-X bonds, the primary substrate
specificity of ICE itself, suggesting that conversion of p45 to its
active heterodimeric form might be an autocatalytic event(17) ,
that is, the result of the enzyme activity of ICE itself. The Asp
residues at postions 103 and 297 in the ICE precursor are conserved in
Ced-3 (residues 131 and 371 of the putative p56 Ced-3
zymogen)(6) , suggesting that the active form of the Ced-3
protein might also be generated by autocatalysis.
We performed the studies presented here to determine protease activity of Ced-3 protein and its functional similarities to ICE. To this end, we developed an assay to detect autocatalysis of in vitro translated Ced-3 protein and used this assay together with various substrate and inhibitor studies to demonstrate that Ced-3 is an Asp-dependent cysteine protease.
The various S-labeled proteins were made by using a coupled
transcription and translation (TNT) system (Promega, Madison, WI).
Briefly, 1 µg of DNA was added directly to TNT rabbit reticulocyte
lysate, and reactions were carried out at 30 °C. For some
experiments (see Fig. 3)
S-labeled and nonlabeled
wild-type (WT) Ced-3 proteins were synthesized in parallel reactions.
The nonlabeled WT Ced-3 proteins were mixed with
S-labeled
C364S Ced-3 mutant or human and murine proIL-1
or human PARP at a
ratio of 1:1. Control samples of
S-labeled WT Ced-3 were
mixed with fresh rabbit reticulocyte lysates at a 1:1 ratio to account
for the dilution factor.
Figure 3:
Enzymatic activities of in vitro transcribed and translated p56 CED-3 and the products of its
autocatalysis. A, WT Ced-3 (radiolabeled (lane 1) and
nonradiolabeled) and C364S Ced-3 mutant (radiolabeled (lane
2)) proteins were synthesized in in vitro transcription
and translation reactions. The radiolabeled WT Ced-3 (lane 3)
and C364S mutant (lane 4) proteins were incubated overnight at
room temperature. The radiolabeled C364S Ced-3 mutant protein was also
incubated overnight with nonradiolabeled WT Ced-3 protein (30`
reaction, lane 5)), with nonradiolabeled WT Ced-3 (90`
reaction, lane 6), or with rICE (lane 7). The
molecular weight markers are depicted on the left. The arrows on the right depict the molecular sizes of the
proteolytic fragments. B, radiolabeled human and murine
proIL-1 proteins were synthesized in in vitro transcription and translation reactions (lanes 1 and 4). These proteins were incubated overnight with
nonradiolabeled WT Ced-3 protein (30` reaction, lanes 2 and 5)), or with nonradiolabeled WT Ced-3 protein (90` Ced-3
reaction, lanes 3 and 6)). Positions of 31-kDa
proIL-1
(pIL-1
), the 28-kDa intermediate (28 kDa), and the
17-kDa mature IL-1
(mIL-1
) are indicated with arrows. C, in vitro transcribed and
translated radiolabeled PARP protein (lane 1) was incubated
with nonradiolabeled WT Ced-3 protein (30` reaction lane 2) or
WT Ced-3 protein (90` reaction, lane 3). Positions of
full-length PARP and the 85- and 24-kDa proteolytic fragments are
indicated by arrows.
Figure 1: Kinetics of p56 Ced-3 protein synthesis and degradation after the initiation of in vitro transcription and translation reaction. Samples were removed to denaturing buffer at the indicated time points and analyzed at the completion of the experiment on a 10-20% Tris-Tricine gradient polyacrylamide gel (Integrated Separation Systems). The molecular weight size markers are indicated on the left. The arrows on the right indicate the various lower molecular weight degradation products of the p56 Ced-3 protein.
To determine whether the breakdown
of the in vitro translated p56 Ced-3 protein, as shown in Fig. 1, was the result of autocatalysis of Ced-3 or an activity
characteristic of the lysates, we performed the following experiments.
Using the in vitro TNT reactions, we made Ced-3, human p45
ICE, and human proIL-1 proteins and incubated these proteins in
the rabbit reticulocyte lysates overnight at room temperature in order
to determine their stability in this assay system. The results in Fig. 2show that unlike Ced-3, the p45 ICE and proIL-1
proteins were stable. The addition of recombinant ICE (derived from sf
9 cells infected with baculovirus encoding the ICE protein) resulted in
the processing of the in vitro translated p45 ICE and
proIL-1
proteins to their respective cleavage products (Fig. 2). The degradation of the p45 ICE and proIL-1
proteins, shown in Fig. 2, was therefore the result of the
activity provided by active ICE, not by a nonspecific activity found in
the lysates. One can infer, therefore, that the observed degradation of
Ced-3 also results from specific proteolysis rather than a nonspecific
activity.
Figure 2:
Human p45 ICE and human proIL-1
proteins made in the in vitro transcription and translation
reactions do not undergo degradation. A, human p45 ICE protein
incubated overnight at room temperature in the absence (lane
1) or presence (lane 2) of rICE. B, human
proIL-1
protein incubated overnight at room temperature in the
absence (lane 3) or presence (lane 4) of rICE. C, Ced-3 protein incubated overnight at room temperature in
the absence of rICE (lane 5). The arrows on the left indicate the various molecular species
generated.
To demonstrate that the degradation of p56 Ced-3 protein was due to the proposed protease activity of Ced-3 (autocatalysis), we used a mutant called the C364S mutant, in which the putative active site cysteine, residue 364, was replaced by a serine. The results in Fig. 3A show that the TNT-synthesized C364S mutant was stable and did not generate the lower molecular weight bands, while the WT p56 Ced-3 protein did degrade, as evidenced by the presence of lower molecular weight bands. These data demonstrate that p56 Ced-3 protein degrades by autocatalysis and that cysteine 364 plays a crucial role in this proteolytic process.
To determine if WT p56 Ced-3 can cleave the C364S mutant and if the products of p56 Ced-3 autocatalysis possess proteolytic activity, WT p56 Ced-3 protein (30-min TNT reaction) or products of p56 Ced-3 autocatalysis (90-min TNT reaction) were mixed with the C364S mutant protein and incubated overnight at room temperature. Both the 30- and 90-min Ced-3 reaction products cleaved the C364S mutant protein, as demonstrated by the appearance of lower molecular weight bands shown in Fig. 3A. Cleavage of C364S mutant by WT p56 Ced-3 or by the autocatalysis products of p56 Ced-3 generated the same sized bands as did autocatalysis of WT Ced-3. Collectively, these data demonstrate that the products of p56 Ced-3 autocatalysis retain proteolytic activity. Recombinant ICE (produced by baculovirus-infected sf 9 cells) also cleaved the C364S mutant protein (Fig. 3A).
Figure 4:
Inhibition of p56 Ced-3 autocatalysis. A, protease inhibition profile of p56 Ced-3 autocatalysis; B, effects of anti-apoptosis compounds on p56 Ced-3
autocatalysis. Wild-type p56 Ced-3 protein was made in a 30` in
vitro transcription and translation reaction. Aliquots of p56
Ced-3 protein were incubated overnight at room temperature in the
absence or presence of various test compounds. Only the p56 band is
shown for each lane. C, effects of various compounds at
blocking autocatalytic cleavages of p56 Ced-3. Except for iodoacetamide
(1000 µM), all compounds were tested at a concentration of
100 µM. D, the ability of proIL-1- and
PARP-derived tetrapeptide aldehyde inhibitors to block autocatalytic
cleavages of p56 Ced-3. Leupeptin was used as an aldehyde control. All
peptides were tested at a concentration of 100 µM. The arrows on the right indicate the molecular weights of
autocatalytic fragments of p56 Ced-3.
We also determined if all the autocatalytic cleavages within the Ced-3 molecule are equally sensitive to various inhibitors. The results in Fig. 4C demonstrate that ATA and iodoacetamide blocked all autocatalytic cleavages of the p56 Ced-3 for up to 16 h of incubation at room temperature. In contrast and as observed in Fig. 4, A and B, E-64 and pepstatin were completely ineffective at inhibiting Ced-3 autocatalysis.
Since in vitro transcribed and translated PARP protein was cleaved
more efficiently by Ced-3 than proIL-1 (Fig. 3), we tested
PARP-derived peptide (Ac-DEVD-CHO; a potent inhibitor of CPP32) and
proIL-1
-derived peptide (Ac-YVAD-CHO; a potent inhibitor of ICE)
for their abilities to block Ced-3 autocatalysis. The results in Fig. 4D demonstrate that the two peptide aldehydes
inhibited Ced-3 autocatalysis to varying degrees. Ac-DEVD-CHO blocked
all autocatalytic cleavages of p56 Ced-3 for up to 16 h of incubation,
whereas, in the presence of Ac-YVAD-CHO, some p56 autocatalysis was
observed at 16 h postincubation. The control peptide aldehyde
Ac-LLR-CHO (leupeptin) was ineffective at blocking Ced-3 autocatalysis.
The data in this report demonstrate the protease nature of in vitro transcribed and translated Ced-3 protein. 1) In
vitro translated p56 Ced-3 was rapidly degraded in our assay
system, while other proteins were not, suggesting autocatalytic
activity of Ced-3. 2) Replacement of the putative active site cysteine
with serine (C364S mutant) completely abolished Ced-3 autocatalysis. 3)
WT p56 Ced-3 and products of its autocatalysis cleaved the C364S mutant
protein, generating proteolytic fragments of the same size as the WT
p56 autocatalysis fragments. 4) Autoprocessed Ced-3 also cleaved
certain of the ICE-sites in human and mouse proIL-1, although it
was far less effective than ICE with these substrates. 5) WT p56 Ced-3
and the products of its autocatalysis cleaved human PARP as well.
Collectively, these observations clearly demonstrate that both the p56
and autoprocessed forms of Ced-3 are proteases.
We found an unusual
inhibition profile of Ced-3 autocatalysis using various classes of
protease inhibitors, similar to the profile previously demonstrated for
ICE(17, 18) . All active site protease inhibitors
reported to inhibit ICE activity (18) also inhibited Ced-3
autocatalysis, while E-64, an inhibitor of many cysteine proteases, did
not inhibit ICE activity (18) or Ced-3 autocatalysis. The
three-dimensional structure of human ICE in complex with the ICE
inhibitors Ac-YVAD-chloromethylketone (19) or Ac-YVAD-aldehyde (20) revealed that, in the crystalline state, ICE is a
homodimer ((p20/p10)) of p20/p10 heterodimers, with each
p20/p10 subunit within the homodimer containing an active site. The
catalytic cysteine and histidine residues as well as the residues
defining the P1 carboxylate binding pocket of ICE are conserved in
Ced-3(19, 20) , suggesting that mechanistically the
active sites of ICE and Ced-3 are similar. The functional observations
presented here support the above notion. However, only 2 out of 6 amino
acid residues of ICE that interact with the P2-P4 residues of the
tetrapeptide inhibitors are conserved in
Ced-3(6, 19, 20) , suggesting that there
might be differences in substrate binding/recognition. The data
demonstrating a strong correlation between the efficiency of cleavage
of PARP and proIL-1
by Ced-3 and the potency of PARP- and
proIL-1
-derived peptides to inhibit Ced-3 autocatalysis might
reflect such differences in substrate binding/recognition.
Interestingly, 5 out of 6 amino acid residues that interact with P2-P4
residues of the substrate are conserved in Ced-3 and CPP32 and, like
Ced-3, CPP32 cleaves PARP more efficiently than proIL-1
. Moreover,
as with Ced-3, the PARP-derived peptide (Ac-DEVD-CHO) is a more potent
inhibitor of CPP32 than the proIL-1
-derived peptide
(Ac-YVAD-CHO)(12) .
We found that p56 Ced-3 and
autoprocessed Ced-3 showed differences in substrate specificity, as had
previously been demonstrated for p45 ICE and p20/p10
ICE(18, 32, 33) . The p56 form of Ced-3
cleaved the C364S mutant protein but did not cleave human or murine
proIL-1, while autoprocessed Ced-3 cleaved all three substrates.
Similarly, the p45 ICE precursor cleaved the active site cysteine p45
ICE mutant (32) but did not cleave pro-IL-1
(33) ,
while the active p20/p10 form of ICE derived from autocatalysis of the
p45 ICE precursor cleaved both substrates(32, 33) .
These differences in the enzymatic activities of the precursor and the
active forms of enzymes might reflect differences in substrate
specificities and/or specific activities of the different forms of the
enzymes.
Although Ced-3 autoprocessed to lower molecular weight
molecules retains proteolytic activity, we do not know which of the
lower molecular weight fragment(s) constitutes the active Ced-3
component(s). It is possible that the processed form of Ced-3, like
ICE, might function as a hetero/homodimer. The differences in the
catalytic activities of p56 Ced-3 and autoprocessed Ced-3 could result
from conformational changes in and around the active site that might
accompany processing, as occurs in processing and activation of ICE. A
1000-fold excess of Ac-YVAD-chloromethylketone is required to inhibit
the activity of p45 precursor ICE as compared with p20/p10
ICE(32) . Furthermore, in an affinity labeling study using
biotinylated tetrapeptide inhibitor, p45 precursor ICE was labeled with
an EC of 5 µM, and p20/p10 ICE was labeled
with an EC
of 1 nM(34) . These
observations demonstrate that the affinity of ICE for the inhibitor
increased as ICE proceeded from the p45 precursor form to the p20/p10
active form, suggesting that active site conformational changes
occurred during processing. Analogous conformation differences in the
active sites of p56 Ced-3 and autoprocessed Ced-3 might explain the
differences in substrate specificity or differences in specific
activities between these forms of the enzyme.
Although members of
the Ced-3/ICE family of proteases have been implicated in the process
of apoptosis(6, 7, 8) , our recent studies
with ICE-deficient mice demonstrated that ICE per se is not a
critical protease in several apoptosis pathways(35) . We found
no evidence of an apoptosis defect in ICE-deficient mice during
embryogenesis, development of the immune system, in vitro spontaneous apoptosis of thymocytes, dexamethasone and
irradiation-induced apoptosis of thymocytes, or
lipopolysaccharide/ATP-induced apoptosis in peritoneal macrophages (35) . However, ICE may play a role in the apoptosis pathway
triggered via the Fas receptor(36) . Apoptosis induced via the
Fas receptor was blocked by the serpin, crmA(37) , an inhibitor
of ICE and possibly of ICE family members(6, 11) .
Collectively, these studies suggest that ICE per se is not
critically involved in many apoptosis pathways. However, ICE may
function redundantly with other ICE family members, certain of which
may play a role in specific apoptosis pathways.
The involvement of
Ced-3/ICE family of proteases in the process of apoptotic cell death,
raises the possibility that some of the known inhibitors of apoptosis
might interfere with the functioning of these proteases. Of the three
agents we tested, only ATA inhibited Ced-3 autocatalysis, but ATA did
not inhibit ICE activity, suggesting that one of the mechanisms by
which ATA inhibits apoptosis might be its ability to inhibit a
Ced-3/ICE family member involved in an apoptosis pathway, such as
PrICE, Nedd2/Ich-1, Ich-2, CPP32, ICE III, Mch2, or some
as yet unidentified member. Studies are underway to determine if ATA
inhibits any of the other known ICE family member (s).
Since ICE is
absolutely required for the generation of mature, biologically active
IL-1(35, 36) , it may be possible to design
selective inhibitors of ICE to block mature IL-1
production in
order to treat inflammatory diseases without influencing the apoptotic
pathway(s). Conversely, specific inhibitors of ICE family member(s)
involved in apoptosis could be developed for the treatment of
apoptosis-dependent diseases, such as neurodegenerative disorders and
AIDS(38) .
Addendum-Since the submission of this manuscript, a manuscript by Xue and Horvitz has appeared showing protease activity of Ced-3 ((27) ).