(Received for publication, September 12, 1994; and in revised form, December 15, 1994)
From the
To understand the mechanism of interleukin-1 converting
enzyme (ICE) activation in apoptosis, we analyzed the expression of ICE
mRNA in two human cell lines by reverse transcription-polymerase chain
reaction technique. This resulted in the identification and cloning of
four alternatively spliced ICE mRNA isoforms. Although all the
alternative splicing events were within the coding sequence of ICE, the
four ICE isoforms maintained open reading frames and were designated as
ICE
,
,
, and
. In ICE
, most of the propeptide
(amino acids 20-112) is deleted, which suggests that it may
function as a catalyst for ICE autoprocessing in vivo. In
ICE
, amino acids 288-335, which contain the cleavage sites
between the p20 and p10 subunits of ICE, are deleted thus resulting in
its inactivation. Intriguingly, in ICE
amino acids 20-335,
which encompass most of the propeptide and the p20 subunit, are deleted
resulting in the formation of a molecule that is homologous to the p10
subunit. Examination of the ability of these four ICE isoforms to cause
apoptosis revealed that only the parental ICE
and isoforms
and
, but not isoforms
and
, can induce apoptosis when
overexpressed in Sf9 insect cells. In addition, coexpression of the p20
and p10 but not the p20 and ICE
in Sf9 cells results in apoptosis.
Interestingly, expression of ICE
and to a lesser degree ICE
resulted in extension of the survival of baculovirus-infected cells in
a manner similar to expression of BCL2. The ability of ICE
to
extend the survival of Sf9 cells suggests that baculovirusinduced
apoptosis in these cells is mediated by an ICE-like protease. We show
that ICE
can bind to the p20 subunit of ICE and potentially may
compete with the p10 subunit to form an inactive ICE complex.
Therefore, by acting as a dominant inhibitor of ICE activity, ICE
may regulate ICE activation in vivo.
Interleukin-1 converting enzyme (ICE) (
)is a
cytoplasmic cysteine protease that cleaves inactive 31-kDa
pro-IL-1
to generate the active 17.5-kDa proinflammatory cytokine
IL-1
(1, 2) . ICE is expressed in many tissues as
an inactive proenzyme polypeptide of 404 amino acids and a relative
molecular mass of 45 kDa (p45)(3, 4) . Active ICE is
produced after proteolytic cleavage of the proenzyme p45 to generate
two subunits of molecular mass = 20 and 10 kDa, known as p20 and
p10 subunits(3, 4) . Recent crystal structure analysis
of active ICE demonstrated that the two subunits associate with each
other to form a (p20)
/(p10)
tetramer (5) also referred to as a (p20/p10)
homodimer(6) . The structure of ICE is unique and is not
related to any known protein structures, including those of other
cysteine proteases(6) . ICE is also unusual in its substrate
specificity. ICE requires an Asp in the P1 position and a small
preferably hydrophobic residue in the P1`
position(7, 8) . Only the serine protease granzyme B
and its homologs have a similar requirement for Asp in the P1
position(9) . Sequence homology between ICE and the Caenorhabditis elegans cell death gene product CED-3 suggests
that mammalian ICE or its homologs might be involved in apoptosis. The
two proteins share an overall 28% sequence identity (10) . A
43% identity is observed when a region that contains the enzyme active
site is compared(10) . A significant homology between ICE or
CED-3 and a newly discovered mouse protein known as Nedd2 was also
demonstrated in a recent study(11) . The significance of this
homology to CED-3 was demonstrated when overexpression of ICE or Nedd2
in fibroblasts resulted in apoptosis(11, 12) .
Expression of crmA, a poxvirus-specific inhibitor of
ICE(13) , was able to block ICE-induced apoptosis in
fibroblasts and to protect ganglion neurons from apoptosis induced by
nerve growth factor depletion(12, 14) . Therefore, ICE
or ICE homologs may play an important role in apoptosis in vertebrates,
similar to CED-3 in nematodes. In this study we describe the cloning
and characterization of four novel alternatively spliced ICE mRNA
isoforms. The significance of these isoforms with regard to ICE
processing and apoptotic activity is discussed.
Figure 1:
Nucleotide and predicted amino acid
sequence of ICE isoforms. Colinear sequence alignment of ICE cDNA
with ICE isoforms
,
,
, and
cDNAs. The predicted
amino acid sequence of ICE
is shown above the nucleotide
sequence. The pentapeptide containing the ICE active site Cys-285 is boxed. Dotted lines indicate the spliced sequences in
ICE isoforms
,
,
, and
. Amino acid and nucleotide
residues are numbered to the right of each sequence.
The two PCR primers, ICE1 and ICE2 (see ``Materials and
Methods''), used to amplify and clone ICE
and the other ICE
isoforms are indicated by solid
arrows.
Figure 2:
Structure and alternative splicing of ICE
mRNA. The primary structure of human proICE (p45) and the
proteolytically generated ICE subunits p20 and p10 are shown as rectangles. The alternatively spliced ICE mRNA isoforms are
represented by solid bars. All nine exons are indicated by numbers above the solidline representing
ICE mRNA. Alternatively spliced exons in other ICE isoforms are
shown as brokenlines.
Figure 3:
In vitro translation of ICE isoforms. ICE
mRNA isoforms were in vitro translated in the presence of
[S]methionine and then analyzed by SDS-PAGE and
autoradiography as described under ``Materials and Methods.'' Panel A, autoradiogram of
S-labeled ICE
,
,
, and
(lanes1-4). Panel
B,
S-labeled ICE
,
,
, or
(lanes
1-4) or mixtures of ICE
and
(lane 5),
ICE
and
(lane 6), or ICE
and
(lane
7) were incubated at room temperature for 24 h and then analyzed
by SDS-PAGE and autoradiography. Molecular size markers are indicated
on the left of A.
Figure 4:
Effect of ICE isoform expression on the
viability of baculovirus-infected Sf9 cells. Sf9 cells were infected
with wild type baculovirus, recombinant baculoviruses encoding
ICE,
,
,
, or
, or recombinant baculovirus
encoding BCL2. Panels A and B, at the indicated times
post-infection, the viability of baculovirus-infected Sf9 cells was
determined by trypan blue exclusion in a hemocytometer. Panel
C, determination of internucleosomal DNA cleavage. Total cell DNA
was isolated from Sf9 cells expressing ICE
(lane
),
ICE
(lane
), ICE
(lane
),
ICE
(lane
), or ICE
(lane
) at 48
h post-infection and electrophoresed in a 1.8% agarose gel containing
ethidium bromide. DNA isolated from cells infected with the wild type
virus for 48 h was used as a control (laneWT). Lane M, molecular size markers.
Because ICE is homologous to the
p10 subunit of active ICE (Fig. 5), we decided to test whether
its coexpression with the p20 subunit could generate an active ICE
heterodimer. As shown in Table 2, expression of either the p10,
p20, or ICE
in Sf9 cells does not cause apoptosis. On the other
hand, coexpression of the p10 and p20 subunits resulted in apoptosis
within the same time frame as did ICE
. In contrast, coexpression
of ICE
and p20 does not cause apoptosis in Sf9 cells. These
results suggest that the first 19 amino acids of the p10 subunit are
essential for ICE activity. Substitution of these amino acids as in
ICE
may result in loss of activity.
Figure 5:
Predicted amino acid sequence of ICE.
Colinear sequence alignment of ICE
and ICE p10 subunit. The
alignment was made using Telnet 2.4.01 MacTCP program based on the
evolutionary distance between the amino acids (gap weight 3 and gap
length weight 0).
Figure 6:
In vitro interaction of ICE
or p10 with glutathione S-transferase-p20 fusion protein.
Reticulocyte lysate aliquots of in vitro synthesized and
S-labeled ICE p10 subunit (panel A) or ICE
(panel B) were incubated with a glutathione S-transferase-p20 fusion protein immobilized on
glutathione-Sepharose affinity resin (lanes +) or with free
glutathione-Sepharose affinity resin (lanes -) for 1 h at 30
°C. A reticulocyte lysate aliquot containing
[
S]methionine but no mRNA (no translation
control) was also incubated with glutathione S-transferase-p20
fusion protein immobilized on glutathione-Sepharose affinity resin (left lane in A). After the incubation period, the
resins were washed and the bound proteins were analyzed on a
5-20% gradient SDS-polyacrylamide gel. The gel was then stained,
dried, and exposed to x-ray film. Molecular size markers are indicated
on the left of A.
In this report we have identified and characterized four
human ICE mRNA isoforms. These four cDNAs result from one or more
alternative splicing events involving exons 2-7 of the ICE gene (17) (see Fig. 2). In ICE the deletion of exon 3
resulted from splicing of the DNA sequence between exons 2 and 4 using
intron 2 splice donor and intron 3 splice acceptor ( Table 1and (17) ). Similarly, the deletion of exon 7 in ICE
resulted
from splicing of the DNA sequence between exon 6 and 8 using intron 6
splice donor and intron 7 splice acceptor ( Table 1and (17) ). On the other hand, the deletions within exons 2-7
in ICE
,
, and
resulted from the use of an alternative
splice donor located within the coding sequence of exon 2 ( Fig. 1and Table 1). However, in ICE
and
intron
3 splice acceptor was used, whereas in ICE
intron 7 splice
acceptor was used (Table 1). When expressed in Sf9 cells, two of
these isoforms, ICE
and ICE
had similar activity to the
parental ICE
isoform. The deletion of 21 or 93 amino acids from
the N-terminal propeptide in ICE
and ICE
, respectively, had
no effect on their ability to induce apoptosis in Sf9 cells. This is
not surprising since the propeptide is not required for ICE activity.
The ability of these isoforms to cause apoptosis was associated with
complete processing of their respective precursor peptides to the p20
and p10 subunits. Only p20 and p10 subunits but no precursors were
detected in Sf9 cells expressing ICE
,
, or
at 48 h
post-infection (data not shown). This suggests that Sf9 cells may
contain a protease that can cleave proICE, or that proICE itself
possesses an autocatalytic activity. So far the mechanism by which
proICE is post-translationally processed to the active ICE heterodimer
is not known. It is also not clear whether the propeptide influences
the kinetics by which the ICE heterodimer is generated. There is some
evidence that active ICE is generated by an autoprocessing mechanism.
Thornberry et al.(4) demonstrated that purified
active ICE heterodimer can cleave the in vitro translated p45
proICE molecule, to generate several intermediates including the p34,
p20, and p10 forms. They also demonstrated that the p45 proICE is
stable for a prolonged incubation at room temperature, suggesting that
p45 lacks an intramolecular autoprocessing activity(4) . This
is consistent with our findings that in vitro translated
proICE
,
, and
, were stable for a prolonged period of
time. Nevertheless, our in vitro translation reactions seemed
to contain intermediate p34 species in proICE
and
reactions,
similar in size to proICE
, but not in ICE
reaction. This
suggests that proICE
or
may have partial intramolecular
autoprocessing activity and that the first step in autoprocessing of
ICE, is removal of the propeptide. In a recent study, Wilson et al.(5) stated that purified p45, and the p30 (p34 here) form
which lacks the N-terminal propeptide, can autoprocess to the active
form in vitro, by manipulation of the enzyme concentrations
and temperature(5) . They also demonstrated that a mutant p30
(p34 here) in which the active site Cys285 has been changed to Ala was
inactive and was not processed to the p20/p10 heterodimer in
vivo. Therefore, whether active ICE heterodimer is generated by an
intra- or intermolecular processing mechanism, we believe that the
removal of the propeptide sequence may accelerate the autoprocessing
mechanism. The removal of most of the propeptide at the level of mRNA
splicing as in ICE
, may provide a p34 ICE species that can be more
readily converted to active ICE heterodimer than the full-length p45
species. Therefore, ICE
may act as an in vivo catalyst
for generation of active ICE heterodimer. Once an active ICE
heterodimer is generated, it may then act on the p45 species and
convert it to the active form.
Another way by which ICE activity may
be regulated is at the level of formation of ICE heterodimer. The
ICE cDNA codes for a protein which corresponds to the p10 subunit
of ICE except for the first 19 amino acids, which are derived from exon
2 in ICE
, and from exon 7 in the p10 subunit (Fig. 1, Fig. 2, and Fig. 5). We have demonstrated that ICE
,
like the p10 subunit, can form a heterodimer with the p20 subunit. The
crystal structure of ICE complexed to a tetrapeptide aldehyde inhibitor
suggests that the side chains of p10 residues Val-338 to Pro-343
interact with the inhibitor, except for Ser-339(5) . Although
all these residues are present in ICE
, the ICE
/p20
heterodimer is inactive. This could be attributed to the fact that
active ICE exists as a (p20)
/(p10)
tetramer in
which the participation of p10 residues 318-322 in the formation
of this tetrameric complex is essential(5) . Because 4 of these
residues are substituted in ICE
(Fig. 5), this may prevent
the formation of a (p20)
/(ICE
)
tetramer.
The biological significance of the expression of an alternatively
spliced ICE
isoform is realized from its ability to modulate ICE
activity. ICE
might compete with p10 for binding to p20 in
vivo. This could be why overexpression of ICE
in Sf9 cells
resulted in a delay of apoptosis in a fashion similar to or even better
than BCL2 expression. Insect Sf9 cells apparently express an ICE-like
protein which might be involved in insect cell apoptosis. Support for
the existence of an ICE-like molecule in Sf9 cells was obtained from
our overexpression studies of pro-IL-1
. Overexpression of
pro-IL-1
in Sf9 cells resulted in its cleavage to the 17.5-kDa
active IL-1
cytokine. (
)Three ICE mRNA species (2.5,
1.9, and 0.5 kb) have been detected in THP-1 cell line and several
other normal human tissues, including peripheral blood monocytes,
peripheral blood lymphocytes, peripheral blood neutrophils, resting and
activated peripheral blood T-lymphocytes, and placenta(3) . We
believe that the smallest 0.5-kb mRNA is the ICE
isoform. ICE
transcript is highly expressed in peripheral blood neutrophils and
placenta(3) . The significance of this high level of expression
is not yet established. We speculate that, by acting as a dominant
inhibitor, ICE
may inhibit ICE activity thus indirect regulating
apoptosis in these tissues.
Regulation of the biological activity of a protein by alternative splicing is a well known phenomenon(19) . Examples where alternative splicing has contributed to different apoptotic activities are found in the gene products of bclx(20) and grb2(21) . The bclx gene is expressed as two alternatively spliced isoforms, Bclx-l and Bclx-s(20) . The Bclx-s isoform lacks 63 amino acids as a result of internal splicing within the first coding exon of the bclx gene(20) . Whereas Bclx-l protects cells from apoptosis, Bclx-s has an opposite effect(20) . This is probably due to forming of an inactive heterodimer with Bcl2 or Bclx-l. The Grb2 isoform Grb3-3 has a deletion in the Src homology 2 (SH2) domain as a result of alternative splicing of one exon(21) . Grb3-3 has been shown to cause apoptosis in Swiss 3T3 cells by acting as a dominant inhibitor of Grb2 function, probably by forming an inactive heterodimeric complex with other protein partners(21) . In conclusion, the ICE isoforms described here are another example where the biological activity of a protein was altered by alternative splicing. This is an important tissue-specific regulatory mechanism. Deregulation of alternative splicing of the ICE gene in certain tissues could result in abnormal levels of ICE activity. Understanding of this mechanism could contribute significantly to our knowledge of this important enzyme. This is especially important in the treatment of a number of degenerative diseases such as Alzheimer's and Parkinson's diseases, where ICE activity might be elevated(22) .
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U13697[GenBank]-U13700[GenBank].