(Received for publication, November 8, 1995; and in revised form, November 22, 1995)
From the
We have identified and characterized a novel cysteine protease
named CMH-1 that is a new member of the interleukin 1 converting
enzyme (ICE) family of proteases with substrate specificity for
Asp-X. CMH-1 has the highest similarity to CPP32 (52% amino
acid identity) and MCH2 (31% identical). CMH-1 shares conserved amino
acid residues that form the core structure of ICE as well as those
residues involved in catalysis and in the P1 aspartate binding.
Overexpression of CMH-1 in COS cells resulted in the processing of
CMH-1 and the induction of apoptosis of transfected cells. Coexpression
of CMH-1 with poly(ADP-ribose) polymerase (PARP) also resulted in a
specific cleavage of PARP. Purified recombinant CMH-1 cleaved PARP but
not interleukin 1
precursor in vitro.
Programmed cell death (apoptosis) plays an important role in
embryonic development, homeostasis, and in diseases such as
neurodegenerative disorders, autoimmune diseases, and cancer (for
review, see Ellis et al.(1991)). Apoptosis has been
characterized by a set of cellular events including cell shrinkage,
chromatin condensation, and DNA fragmentation. It is becoming apparent
that activation of proteases is a crucial event in the cellular
execution of apoptosis (for review, see Martin and Green(1995)).
Genetic studies in the nematode Caenorhabditis elegans have
provided evidence that the Ced-3 gene is indispensable for
cell death during worm development (Yuan et al., 1993). The
CED-3 protein shares 28% sequence identity with mammalian
interleukin-1 (IL-1
) (
)converting enzyme (ICE)
that cleaves the inactive IL-1
precursor to the proinflammatory
cytokine (Thornberry et al., 1992). ICE and CED-3 are members
of a new cysteine protease family that also includes mammalian enzymes
of Tx, ICE
III, NEDD-2/ICH-1, CPP32, and MCH2 (Faucheu et al., 1995; Munday et al., 1995; Wang et
al., 1994; Kumar et al., 1994; Fernandes-Alnemri et
al., 1994, 1995). These cysteine proteases are composed of two
subunits of approximately 20 kDa and 10 kDa derived from processing of
precursor polypeptides. They share conserved amino acid residues for
catalysis and binding of the P1 aspartate residue (Wilson et
al., 1994; Walker et al., 1994). Overexpression of
cysteine proteases in the Ice/Ced-3 gene family leads
to apoptosis of transfected cells (Miura et al., 1993; Faucheu et al., 1995; Fernandes-Alnemri et al., 1994, 1995;
Gu et al., 1995a; Tewari et al., 1995), and ICE
inhibitors block cell death of neurons deprived of neurotrophic factors
(Gagliardini et al., 1994; Milligan et al., 1995).
ICE or related proteases have also been implicated in Fas- and tumor
necrosis factor
-mediated apoptosis (Kuida et al., 1995;
Enari et al., 1995; Los et al., 1995; Tewari and
Dixit, 1995; Miura et al., 1993), suggesting a physiological
role for these cysteine proteases in apoptosis.
Recently, Nicholson et al.(1995) have identified CPP32 as a cysteine protease that is responsible for the cleavage of poly(ADP-ribose) polymerase (PARP), a DNA repair enzyme that is cleaved from a 116-kDa polypeptide into 31-kDa and 85-kDa polypeptides at the onset of apoptosis and in nuclei treated with apoptotic cytosolic extracts (Kaufmann et al., 1993; Lazebnik et al., 1994). Darmon et al.(1995) found that cytotoxic T lymphocyte-specific granzyme B can process and activate CPP32, resulting in PARP cleavage in target cells, suggesting that granzyme B-induced apoptosis may be mediated by a protease activation cascade involving CPP32. Using polymerase chain reaction, Fernandes-Alnemri et al.(1995) identified a second human Ced-3 homolog, MCH2, that is 38% identical with CPP32. Overexpression of MCH2 in insect cells induces apoptosis, and purified MCH2 cleaves PARP in vitro, suggesting a possible role of MCH2 in vertebrate apoptosis. We recently cloned a cDNA encoding a 34-kDa polypeptide that is highly homologous to CPP32 and Mch2. In this report we describe the cloning, expression, and characterization of this new cysteine protease named CMH-1 (CPP32/Mch2 homolog).
The
full-length cDNA contains a single large open reading frame encoding a
polypeptide of 303 amino acids which we have named CMH-1 (Fig. 1). This protein was found to have the highest similarity
to CPP32 and MCH2 with 52% and 31% identity, respectively. It shares
limited overall similarity with other members of the Ice/Ced-3 gene family, including ICE itself which is
19% identical. However, there is strong similarity between CMH-1 and
ICE in the region that comprises the core of the ICE structure as well
as a number of residues surrounding the active site Cys and His
in ICE (Wilson et al., 1994;
Walker et al., 1994). In particular, all of the CMH-1 residues
which line the putative substrate binding pocket at the P1 position are
identical with those found in ICE, including Arg
and
Arg
, corresponding to ICE Arg
and
Arg
, strongly suggesting that CMH-1 has substrate
specificity for Asp in this position as seen in other members of this
family (Fig. 1B). There is much less sequence
conservation in the binding pockets corresponding to the P3-P4 residues
between CMH-1 and other homologs.
Figure 1:
A,
predicted amino acid sequence of the human Cmh-1. The
conserved QACRG motif is underlined, and the catalytic
residues His and Cys
are shown in bold. The autoprocessing aspartate sites between P21 and P11
subunits are circled. The putative cleavage for removal of
prodomain occurs at Asp
which is boxed. B,
sequence alignment of CMH-1 and human ICE homologs. The sequence of
CMH-1 was aligned to other human ICE homologs. Residues which are
conserved in more than 70% of the homologs are boxed. The
prodomain of ICE and the putative prodomains of Tx,
ICE
III, and Ich1 are represented by dashes. The
catalytic His and Cys are marked by asterisks. The location of
residues which comprise the S1-S4 substrate side-chain binding pockets
are marked by the numbers 1-4.
Figure 2: PARP cleavage and induction of apoptosis by CMH-1 in COS cells. A, expression, autoprocessing, and PARP cleavage by CMH-1. COS cells were transfected with the tagged wild type or an active site mutant (C186S/W232A/R233E) CMH-1 expression plasmid, alone or in combination with a T7-tagged truncated PARP plasmid (Gu et al., 1995b) as indicated. Twenty-four hours later, the cells were harvested, and expressed proteins were analyzed by immunoblotting with an anti-T7 antibody. Molecular mass is indicated in kilodaltons. The unprocessed and the processed CMH-1 polypeptides were indicated along with the uncleaved truncated PARP (PARP(T)) and the tagged N-terminal fragment of cleaved PARP (PARP*). B, induction of apoptosis in transfected cells by CMH-1 as evidenced by internucleosomal DNA fragmentation. COS cells were mock-transfected (lane 1), transfected with the tagged wild type CMH-1 (lane 2) or the mutant CMH-1 (lane 3), and harvested 36 h post-transfection. Chromosomal DNA was extracted from these cells and analyzed on a 1.8% agarose gel containing ethidium bromide. DNA size markers in kilobases are shown in lane MW.
To determine if CMH-1 protein expressed in COS cells may have other detectable protease activity, we co-expressed CMH-1 in COS cells with a truncated form of PARP, a known substrate for CPP32 and other ICE-like proteases both in vivo and in vitro (Nicholson et al., 1995; Gu et al., 1995b). We found most of the 45-kDa PARP polypeptide was cleaved into a 31-kDa peptide when it is co-expressed with CMH-1, similar to the cleavage of PARP by ICE, Tx, and Nedd-2. This cleavage was abolished when the mutant CMH-1 was used, indicating that the cleavage is specific and that Cmh-1 encodes a protease that can either cleave PARP directly or activate an endogenous protease(s) that in turn cleaves PARP. A small amount of PARP cleavage was also observed when COS cells were transfected with PARP alone; this is most likely due to the presence of (an) endogenous ICE-like protease(s) in the cells.
Overexpression of the CMH-1 protein in COS cells also caused cells to round up, a sign of transfected cells undergoing apoptosis. To confirm this, we analyzed chromosomal DNA from cells transfected with the wild type Cmh-1 and found internucleosomal DNA fragmentation (Fig. 2B). DNA fragmentation was absent from mock-transfected cells or cells transfected with the mutant Cmh-1 (Fig. 2B). Thus, like other ICE-like proteases, CMH-1 is capable of inducing apoptosis when overexpressed in cells.
Figure 3:
Purification of recombinant
(His)-tagged CMH-1. (His)
-tagged
CMH-1(Ala
-Gln
) was expressed and purified
from E. coli soluble fractions by Ni-affinity chromatography
as described under ``Materials and Methods.'' Four µl (2
µg of total protein) of purified fraction were electrophoresed on
16% polyacrylamide gel. The proteins were detected either by Coomassie
Blue staining (lane 1) or by immunoblotting with
anti-(His)
-tag antibody (lane 2). N-terminal amino
acid sequencing indicated that the 12-kDa and 11-kDa bands correspond
to CMH-1 polypeptides starting at Ser
and
Ala
, respectively.
Figure 4:
CMH-1 is a cysteine protease that cleaves
PARP in vitro.A, CMH-1 specifically cleaves PARP but
not IL-1 precursor. Left panel,
S-labeled
PARP(T) prepared by in vitro transcription/translation (Gu et al., 1995) was incubated for 30 min at 37 °C with
approximately 3 nM purified CMH-1 or buffer in the presence or
absence of a 0.5 µM concentration of the tetrapeptide
aldehyde inhibitor DEVD-CHO as described under ``Materials and
Methods.'' Cleavage products were analyzed by SDS-PAGE and
fluorography. PARP(T) denotes the uncleaved truncated form of
PARP, and PARP* indicates the primary cleavage products (32
and 12 kDa). Right panel,
S-labeled IL-1
precursor was incubated alone or with purified recombinant ICE (3
nM) or CMH-1 (3 nM) proteins for 30 min at 37 °C
and analyzed by SDS-PAGE and fluorography. Positions of IL-1
precursor (pIL-1
) and the cleavage product (IL-1
) are
indicated. B, effects of protease inhibitors on the
PARP-cleavage activity of CMH-1 in vitro. Purified CMH-1
(
3 nM) was incubated with
S-PARP for 10 min
at 37 °C in the presence of 0.5 µM DEVD-CHO, 1
µM YVAD-CHO, 16 µg/ml crmA, 5 mMN-ethylmaleimide, 5 mM iodoacetamide, 100
µML-1-chloro-3-(4-tosylamido)-7-amino-2-heptanone (TLCK), 100 µML-1-chloro-3-(4-tosylamido)-4-phenyl-2-heptanone (TPCK), 2 µg/ml aprotinin, or 5 mM EDTA as
indicated. Cleavage products were analyzed by SDS-PAGE and
fluorography.
CPP32 has been implicated to play a major role in apoptosis and cytotoxic T lymphocyte-mediated cell killing as well as cellular regulation of sterol metabolism (Nicholson et al., 1995; Darmon et al., 1995; Wang et al., 1995). CMH-1 and CPP32 share high amino acid similarity and conserved S1, S2, and S4 residues in the active site pocket. This sequence conservation might allow these two proteases to share an overlapping substrate profile and to have similar roles in apoptosis and other physiological processes. It is equally likely that CMH-1 protease has different preferred substrates that remain to be identified. The expression and purification of active CMH-1 protease should facilitate the characterization of the enzyme and its substrates.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U40281[GenBank].