(Received for publication, September 27, 1995; and in revised form, November 28, 1995)
From the
Members of the ICE/ced-3 gene family have been
implicated as components of the cell death pathway. Based on
similarities with the structural prototype
interleukin-1-converting enzyme (ICE), family members are
synthesized as proenzymes that are proteolytically processed to form
active heterodimeric enzymes. In this report, we describe a novel
member of this growing gene family, ICE-LAP3, which is closely related
to the death effector Yama/CPP32/Apopain. Pro-ICE-LAP3 is a 35-kDa
protein localized to the cytoplasm and expressed in a variety of
tissues and cell lines. Overexpression of a truncated version of
ICE-LAP3 (missing the pro-domain) induces apoptosis in MCF7 breast
carcinoma cells. Importantly, upon receipt of a death stimulus,
endogenous ICE-LAP3 is processed to its subunit forms, suggesting a
physiological role in cell death. This is the first report to
demonstrate processing of a native ICE/ced-3 family member
during execution of the death program and the first description of the
subcellular localization of an ICE/ced-3 family member.
Apoptosis, or programmed cell death, is essential for the development and homeostasis of multicellular organisms(1) . It is an active form of cellular suicide encoded by an endogenous program that can be triggered by either internal or external cues. The morphological alterations of programmed cell death include cellular shrinkage, membrane blebbing, and chromatin condensation(2) . Derangements of apoptosis contribute to the pathogenesis of several human diseases including cancer, acquired immunodeficiency syndrome, and neurodegenerative disorders(3, 4, 5) .
Despite its biological importance, the molecular mechanism behind apoptosis remains to be defined. Recently, systematic genetic analysis of Caenorhabditis elegans has identified three genes (ced-3, ced-4, and ced-9) that are important in the regulation of nematode cell death. The proteins encoded by ced-3 and ced-4 are required for all somatic cell deaths that occur during the development of C. elegans(6) . Mutations of ced-3 and ced-4 abolish the apoptotic capability of cells that normally die during development(7) . By contrast, ced-9, which encodes a protein that is homologous to the mammalian gene bcl-2, functions to suppress somatic cell death in the nematode(8) . The function of ced-9 can be partially substituted by ectopic expression of bcl-2 (9). These results suggest that components of the apoptotic machinery are conserved throughout evolution and that mammalian counterparts of ced-3 and ced-4 may play an important role in the mammalian cell death pathway. To date, no homologues of ced-4 have been identified. By contrast, numerous relatives of ced-3 have been characterized, comprising a new gene family of cysteine proteases.
The first mammalian homologue of ced-3 identified was interleukin-1-converting enzyme (ICE) (
)(10) , a cysteine protease involved in the
processing and activation of pro-interleukin-1
to an active
cytokine(11, 12) . Overexpression of ICE or Ced-3 in
Rat-1 cells induced apoptosis, suggesting that ICE may act as the
functional mammalian homologue of Ced-3(13) . However, later
studies refute this possibility, since ICE-deficient mice develop
normally and express no overt defects in apoptosis, except possibly in
the Fas pathway(14, 15) . Furthermore, apoptotic
extracts prepared from chicken DU249 cells failed to cleave the primary
substrate of ICE, pro-interleukin-1
(16) . Instead, an
ICE-like activity in these extracts, termed prICE, cleaved the nuclear
enzyme poly(ADP-ribose) polymerase (PARP) into characteristic
fragments(16) . Purified ICE failed to cleave
PARP(16, 17) , suggesting that prICE was distinct from
ICE. These observations, along with others, suggest that an ICE-like
enzyme, rather than ICE itself, plays a role in the mammalian cell
death pathway.
Five members of the ICE/Ced-3 family have been recently identified and include Nedd-2/ICH1(18, 19) , Yama/CPP32/Apopain(17, 20, 21) , TX/ICH2/ICE rel-II(22, 23, 24) , ICE rel-III(22) , and Mch2(25) . All family members share sequence homology with ICE/Ced-3 and contain an active site QACRG pentapeptide in which the cysteine residue is catalytic. Overexpression of these proteins in a variety of cells causes apoptosis.
Among the ICE/Ced-3 family members thus far cloned, evidence is growing that Yama may act as a distal effector of the apoptotic machinery. Yama has been shown to cleave the death substrate PARP, in addition to being inhibitable by the cowpox serpin, CrmA(17) . Activated Yama (or Apopain), comprised of p17 and p12 subunits, was purified from cell extracts using a tetrapeptide aldehyde inhibitor corresponding to amino acids at the PARP cleavage site(21) . Depletion of Yama from these extracts abrogated their apoptotic potential in vitro(21) . This apoptotic activity could be restored by adding back purified Yama to the depleted extracts(21) . Though the evidence is compelling that Yama serves as a functional mammalian homologue of Ced-3, one cannot exclude a role for other ICE/Ced-3 family members in the cell death pathway. For instance, Yama may be the distal effector of a proteolytic cascade that is comprised of (or activated by) other related family members. A precedent for this is the activation of Yama by purified ICE or granzyme B in vitro(17, 26) . Alternatively, there may exist a redundant cell death pathway in which individual ICE/Ced-3 family members play a role.
Here we report the cloning and characterization of a novel member of the ICE/Ced-3 family designated ICE-LAP3 (for ICE-Like Apoptotic Protease 3) that is closely related to the death effector Yama. ICE-LAP3 is expressed in a variety of tissues and cell lines. Overexpression of ICE-LAP3 in MCF7 cells induces cell death, and mutation of the putative catalytic cysteine residue abolishes its apoptotic potential. Stimulation of the cytokine death receptors, Fas/APO-1 or TNFR-1, triggers processing of pro-ICE-LAP3 to active p20 and p12 subunits. Taken together, our results suggest that ICE-LAP3 is likely a cysteine protease that may have a role in cytokine-mediated cell death.
MCF7 cells and derived transfectants were plated in 150-mm dishes and grown to 80% confluency. Cells were treated with TNF (40 ng/ml) for 22 h, harvested by scraping, recovered by centrifugation, washed once with PBS, and lysed as above.
Figure 2:
ICE-LAP3 expression and subcellular
localization. A, a human adult and fetal tissue
poly(A) Northern blot (Clontech) was probed with
P-labeled ICE-LAP3 cDNA. PBL, peripheral blood
leukocyte. B, a variety of human cell lines were examined for
expression of endogenous ICE-LAP3 protein. Cell lysates were
immunoprecipitated (IP) with anti-ICE-LAP3 antibody (Y) or control pre-immune serum (C) and immunoblotted
with anti-ICE-LAP3 antibody. C, immunolocalization of ICE-LAP3
using the anti-LAP3 polyclonal antisera. Anti-LAP3 reactivity was
visualized with fluorescein isothiocyanate-conjugated anti-rabbit
secondary antibody. Jurkat T cells were stained with peptide-blocked
anti-LAP3 serum (left) and anti-LAP3 serum (right).
Figure 1:
Sequence analysis of ICE-LAP3. A, deduced amino acid sequence of ICE-LAP3. The conserved
pentapeptide QACRG is boxed. Putative Asp cleavage sites are
indicated with an asterisk. The underlined sequence
corresponds to the synthetic peptide used to generate anti-ICE-LAP3
antibody. B, sequence alignment of known members of the human
ICE/Ced-3 family and the nematode gene ced-3. The pentapeptide
QACRG is boxed. Based on the x-ray crystal structure of ICE,
the residues involved in catalysis and substrate binding are shown in color, and the numbers correspond to the position of
residues within ICE. Red-filled circles are catalytic; green-filled triangles represent the binding pocket for the
carboxylate of the P Asp; blue-filled squares indicate the residues adjacent to the P
-P
amino acids. C, phylogenetic analysis of the
ICE/ced-3 gene family.
Yama is an Asp-specific cysteine protease responsible for the
cleavage of the death substrate PARP(17, 21) . The
full-length p32 form, or Pro-Yama, requires proteolytic processing to
active p17 and p12 subunits(21) . The majority of sequence
homology between Yama and ICE-LAP3 lies within a region of ICE-LAP3
that corresponds to the active subunits of Yama. Comparison with Yama
would suggest that ICE-LAP3 contains an N-terminal aspartate cleavage
site and an internal cleavage site defining two subunits that are not
separated by a linker peptide (Fig. 1A). The QACRG
pentapeptide, conserved in all family members, is likewise conserved in
ICE-LAP3. In addition, based on the x-ray crystal structure of
ICE(35, 36) , the amino acid residues
His, Gly
, and Cys
of ICE are
involved in catalysis, while the residues Arg
,
Gln
, and Arg
form a binding pocket for the
carboxylate side chain of the P
aspartic acid. These six
amino acids are conserved in all ICE/Ced-3 family members including
ICE-LAP3 (Fig. 1B). However, residues that form the
P
-P
binding pockets are not widely
conserved among family members, suggesting that they may determine
substrate specificity.
Phylogenetic analysis of the ICE/ced-3 gene family revealed three subfamilies (Fig. 1C). Yama/CPP32/Apopain, ICE-LAP3, and Mch2 are closely related to C. elegans Ced-3 and comprise the Yama subfamily. ICE and the ICE-related genes, TX/ICE rel II/ICH2 and ICE rel III, form the ICE subfamily, while ICH1 and its mouse homologue, Nedd-2, form the Nedd-2 subfamily.
Figure 3:
ICE-LAP3 induces apoptosis and is
activated during Fas- and TNF-induced cell death. A, MCF7
cells were transiently transfected with the reporter gene
-galactosidase and either the truncated form of ICE-LAP3 (p28) or
the mutant version with the catalytic cysteine residue inactivated
(ICE-LAP3 mutant), ICE, or a vector control as described under
``Materials and Methods.'' Percent apoptotic cells represents
the mean value from three independent experiments (mean ± S.D.). B, transfected cells were stained with
5-bromo-4-chloro-3-indoyl
-D-galactoside and examined by
phase contrast microscopy. C, a time course of pro-ICE-LAP3
proteolytic processing generating an active p20 subunit during
Fas-induced apoptosis of BJAB and Jurkat cells. 5
10
BJAB or Jurkat cells were treated with anti-Fas antibody (100
ng/ml) plus protein A (10 µg/ml) for the indicated time periods,
and cell lysates were analyzed by immunoprecipitation and Western blot
analysis with anti-ICE-LAP3 antiserum. D, activation of
ICE-LAP3 during anti-Fas and TNF treatment is blocked by CrmA. Cells
were treated with anti-Fas antibody or TNF and prepared as in C. Left panel, 5
10
BJAB cells or
derived transfectants were treated with anti-Fas antibody (100 ng/ml)
plus protein A (10 µg/ml) for 6 h. Right panel, MCF7 or
derived transfectants were treated with TNF (40 ng/ml) for 22
h.
In conclusion, we have identified a novel death-inducing protein of the ICE/Ced-3 family. ICE-LAP3 was immunolocalized to the cytoplasm, where the death effector machinery is thought to reside(37) . This is the first report describing the activation of an endogenous ICE/Ced-3 cysteine protease during Fas- and TNF-induced cell death, suggesting that it is a mediator of the cytokine-mediated cell death pathway. Further, there is compelling evidence that a family member, Yama, acts as a distal effector of the cell death pathway(17, 21) . The high homology between Yama and ICE-LAP3 is intriguing and raises many important questions about a potential role of ICE-LAP3 in a general cell death program. One possibility is that ICE-LAP3 also acts as an effector in a redundant cell death pathway distinct from the one utilizing Yama. Alternatively, ICE-LAP3 could act as an upstream enzyme, or ``Yama convertase,'' responsible for the processing of Pro-Yama to an active death protease. The cloning and characterization of the Yama-related protein ICE-LAP3 will help address these specific questions and, in general, enhance our understanding of the cell death machinery and the proteases that compose it.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U39613[GenBank].