©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Cloning and Expression of Four Novel Isoforms of Human Interleukin-1 Converting Enzyme with Different Apoptotic Activities (*)

(Received for publication, September 12, 1994; and in revised form, December 15, 1994)

Emad S. Alnemri (§) Teresa Fernandes-Alnemri Gerald Litwack (§)

From the Department of Pharmacology and the Jefferson Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

To understand the mechanism of interleukin-1beta 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 ICEbeta, , , 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 ICEalpha and isoforms beta 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.


INTRODUCTION

Interleukin-1beta converting enzyme (ICE) (^1)is a cytoplasmic cysteine protease that cleaves inactive 31-kDa pro-IL-1beta to generate the active 17.5-kDa proinflammatory cytokine IL-1beta(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)(2)/(p10)(2) tetramer (5) also referred to as a (p20/p10)(2) 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.


MATERIALS AND METHODS

Cloning of ICE Isoforms

The cDNAs for individual ICE isoforms were cloned by a combination of reverse transcription and polymerase chain reaction techniques (RT-PCR). Reverse transcription was performed on poly(A) RNA from the human T-lymphocyte cell line Jurkat or total RNA from the human monocyte cell line THP-1 using a synthetic primer (ICE-RT) derived from the 3` untranslated sequence of human ICE (3, 4) and Moloney murine leukemia virus reverse transcriptase. The reverse transcription products were then used as templates for PCR using two nested ICE-specific primers ICE1 and ICE2 (see Fig. 1). Primer sequences were as follows: ICE-RT, CAGAACGATCTCTTCAC; ICE1, ATGGCCGACAAGGTCCTG; ICE2, CCTGCCCGCAGACATTCA. The amplified DNA was blunt-ended with T4 DNA polymerase, phosphorylated with T4 polynucleotide kinase, and then fractionated on low melting point agarose gels. Several DNA fragments ranging in size from 0.3 to 1.3 kb were observed after staining the gel with ethidium bromide. After confirming that all these DNA fragments contain ICE sequences by Southern blot analysis with an ICE-specific probe, they were excised from the gel and cloned into a SmaI-cut pBluescript II KS vector (Stratagene). The cloned cDNAs were then sequenced with T3 and T7 sequencing primers and other ICE-specific primers. The largest clone corresponding to the published ICE sequence (3, 4) was designated as KS-ICEalpha. The other ICE clones were designated as KS-ICEbeta, KS-ICE, KS-ICE, and KS-ICE based on decreasing size.


Figure 1: Nucleotide and predicted amino acid sequence of ICE isoforms. Colinear sequence alignment of ICEalpha cDNA with ICE isoforms beta, , , and cDNAs. The predicted amino acid sequence of ICE alpha 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 beta, , , 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 alpha and the other ICE isoforms are indicated by solid arrows.



Construction of Plasmids, Transfer Vectors, and Recombinant Baculoviruses

As outlined above, cDNAs of all ICE isoforms were cloned in the SmaI site of pBluescript II KS plasmid vector under the T7 promoter. The cDNAs for the p20 and p10 subunits were obtained by PCR using synthetic primers (ICE1 and p20TAA for p20; p10ATG and ICE2 for p10) and KS-ICE as a template. The sequence of the p20TAA primer is GGTTTCCAGAAACTCCTACTTAATC and the p10ATG primer is ATGGCTATTAAGAAAGCCCACATA. The amplified DNA fragments were blunt-ended, phosphorylated, and then cloned in the SmaI site of pBluescript II KS vector under the T7 promoter and designated as KS-p10 and KS-p20. The cDNA fragments corresponding to ICEalpha, beta, , , , p20, and p10 were excised from their corresponding Bluescript vectors with BamHI and EcoRI restriction enzymes and subcloned into a BamHI/EcoRI-cut pVL1393 to generate the corresponding recombinant transfer vectors. These recombinant transfer vectors were then used to generate recombinant baculoviruses as described previously(15, 16) .

In Vitro Transcription and Translation

The five KS-ICE or KS-p10 vectors were linearized with EcoRI and then used as templates for T7 RNA polymerase. The in vitro synthesized mRNA was precipitated by ethanol, dried and dissolved in TE buffer (10 mM Tris, pH 8.0, 1 mM EDTA). In vitro translation was performed in rabbit reticulocyte lysate in the presence of 80 µCi/ml [S]methionine and 200 µg/ml in vitro transcribed mRNA. The translation products were then analyzed by SDS-polyacrylamide gel electrophoresis and autoradiography.

Expression of Glutathione S-Transferase-p20 Fusion Protein in Bacteria

The p20 cDNA was subcloned in-frame into the BamHI site of the bacterial expression vector pGEX-5X-3 (Pharmacia Biotech Inc.). The p20 cDNA was obtained by PCR using synthetic primers p20GEX and p20TAA and KS-ICE as a template. The sequence of p20GEX primer is GGGATCCTGAACCCAGCTATGCCCACATCC. The amplified DNA fragment was blunt-ended, phosphorylated, and then cloned in the SmaI site of pBluescript II KS vector under the T7 promoter. The cDNA fragment was then excised from the Bluescript vectors with BamHI and then subcloned in a BamHI-cut pGEX-5X-3. The expression plasmid PGEX-p20 was introduced into E. coli DH5alpha, and protein expression was induced with isopropyl-1-thio-beta-D-galactopyranoside.

Analysis of p20 Interaction with p10 and ICE

Exponentially growing bacteria carrying the expression plasmid PGEX-p20 was induced with isopropyl-1-thio-beta-D-galactopyranoside for 3 h and then lysed by sonication. The recombinant glutathione S-transferase-p20 present in the bacterial lysate was adsorbed to glutathione-Sepharose resin for 10 min at room temperature and then washed three times with phosphate-buffered saline. The immobilized p20 fusion protein was incubated with reticulocyte lysates containing S-labeled p10 or ICE for 1 h at 30 °C and then washed five times with phosphate-buffered saline containing 0.1% Triton X-100. Laemmli sample buffer was then added to the resin, and the eluted proteins were analyzed on a 5-20% SDS-polyacrylamide gradient gel and visualized by autoradiography.


RESULTS

Cloning of Four Novel ICE Isoforms

Employing RT-PCR to analyze the expression of ICE mRNA in the human Jurkat T-lymphocyte cell line, several DNA products of 1248, 1185, 969, 825, and 300 bp were detected by ethidium bromide staining. The two largest PCR products were the most abundant. All five products were detected by Southern blot hybridization using the full-length ICE cDNA as a probe (data not shown). This pattern of RT-PCR products was also observed using total RNA from the human acute monocytic leukemia cell line THP-1. Subsequently, the two largest PCR products (1185 and 1248 bp) were cloned from Jurkat mRNA and the three smaller less abundant products (300, 825, and 969 bp), which represent 10-15% of the larger products, were cloned from THP-1 mRNA. All cloned PCR products were then sequenced (Fig. 1). The largest form (1248 bp) corresponds to the sequence of the full-length ICE as reported previously (3, 4) and was designated as ICEalpha. Intriguingly, sequence analysis of the four smaller PCR products revealed that these forms were generated by independent alternative splicing events of the parental ICE mRNA transcript. Table 1lists the splice donor and acceptor sequences of the human ICE gene (17) that are utilized to generate the four ICE isoforms. All the splice junction sequences within the splice donor and acceptor sites conform to the consensus GT/AG rule (Table 1). The 1185-bp cDNA, designated as ICEbeta, lacks the entire exon 3 of the ICE gene (bp 275-338; amino acids 92-112) ( Fig. 1and Fig. 2). The 969-bp cDNA, designated as ICE, lacks most of exon 2 and the entire exon 3 (bp 59-338; amino acids 20-112). The 825-bp cDNA, designated as ICE is similar to ICE but also lacks the entire exon 7 (bp 863-1006; amino acids 288-335). The smallest 300-bp cDNA, designated as ICE lacks most of exon 2 and exons 3 to 7 (bp 59-1006; amino acids 20-335). All these alternatively spliced ICE isoforms maintained an open reading frame, which gave rise to different translation products (Fig. 3A). ICE proenzyme (ICEalpha here) requires proteolytic cleavage to generate p20 and p10 subunits for enzymatic activation(4) . It is not yet clear whether ICE activation occurs via an intramolecular autoprocessing mechanism or requires limited proteolysis by an unknown protease(4) . However, it has been shown that active ICE heterodimer can cleave reticulocyte lysate translated proICE to generate the p20 and p10 subunits(4) . Because ICE isoforms beta, , and contain the active site Cys-285 but lack part or most of the 119 N-terminal propeptide it was interesting to determine whether these isoforms possess autoprocessing activity. ICE isoforms alpha, beta, , and were in vitro transcribed and translated in rabbit reticulocyte lysates. Translation of ICE mRNA isoforms gave rise to 30-48-kDa translation products (Fig. 3A). We also detected a major 34-kDa product in ICEalpha and ICEbeta translation reactions (Fig. 3A, lanes alpha and beta). This p34 product is similar in size to the ICE translation product (lane ) and could be a processed ICEalpha and beta. However, all these products were stable for 24 h in reticulocyte lysates at room temperature (Fig. 3B). No decrease in the intensity of the 30-48-kDa translation products or appearance of a p20 species was observed after this prolonged incubation (Fig. 3B, lanes alpha-). In addition, incubation of ICEalpha with ICE or ICE, or incubation of ICE with ICE for 24 h at room temperature did not result in processing of these isoforms to the p20 and p10 subunits (Fig. 3B). The stability of the ICE isoforms suggests that ICE isoforms do not possess autocatalytic activity under these in vitro conditions.




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 ICEalpha 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 ICEalpha, beta, , and (lanes1-4). Panel B, S-labeled ICEalpha, beta, , or (lanes 1-4) or mixtures of ICEalpha and (lane 5), ICEalpha 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.



Determination of the Apoptotic Activity of ICE Isoforms

To determine the ability of the individual ICE isoforms to induce apoptosis, each ICE isoform was overexpressed in Sf9 cells with the baculovirus system. The viability of infected cells was determined at various times post-infection. As shown in Fig. 4A, the viability of Sf9 cells infected with the recombinant ICEalpha baculovirus decreased sharply 24-48 h post-infection. Similar effects were also observed with ICEbeta and ICE (Fig. 4B). In contrast, cells infected with the wild type virus, ICE, ICE or BCL2 baculoviruses showed very little decrease in viability during this period (Fig. 4, A and B). The decrease in viability of cells infected with the recombinant ICEalpha, beta, and baculoviruses was due to induction of apoptosis when the recombinant ICE proteins started to rise 24-48 h post-infection. This period is the time at which proteins under the polyhedrin promoter are induced. During this period, cells expressing ICEalpha, beta, or showed characteristic signs of apoptosis including cytoplasmic membrane blebbing, nuclear condensation, and internucleosomal DNA cleavage (Fig. 4C, lanes alpha, beta, and ). These apoptotic signs were not observed in Sf9 cells infected with ICE or baculoviruses (Fig. 4C, lanes and ) or with the wild type (Fig. 4C, lane WT) or BCL2 baculoviruses(18) . In fact, it was surprising to discover that expression of ICE and to a lesser degree ICE conferred some protection and delayed the apoptotic response to baculovirus infection (Fig. 4A). In this experiment, cells infected with ICE baculovirus showed a lesser decrease in viability compared to cells infected with the wild type virus (Fig. 4A). This protective effect was similar to that observed with the expression of BCL2 in these cells (Fig. 4A and (18) ). Coexpression of ICEalpha with BCL2 or ICE resulted in a slight delay in the onset of apoptosis, but was not protective. Because of the lethality of ICEalpha, we detected a significantly lower expression of BCL2 or ICE in this coexpression experiment (data not shown).


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 ICEalpha, beta, , , 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 ICEalpha (lane alpha), ICEbeta (lane beta), 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 ICEalpha. 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).





Determination of the Ability of ICE to Interact with p20

To determine whether ICE can interact with p20, ICE was in vitro transcribed and then translated in reticulocyte lysate in the presence of [S]methionine. The labeled ICE was then incubated with a glutathione S-transferase-p20 fusion protein expressed in bacteria and immobilized on glutathione-Sepharose resin. The p10 subunit was also labeled in reticulocyte lysate with S and used as a control. As shown in Fig. 6, both p10 and ICE were able to interact with the glutathione S-transferase-p20 fusion protein (lanes +). A small amount of p10 and ICE were bound nonspecifically to free glutathione-Sepharose resin (lanes -). These results suggest that ICE can form a heterodimer with p20 and may regulate its activity in vivo by forming an inactive complex.


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.




DISCUSSION

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 ICEbeta 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, ICEbeta and ICE had similar activity to the parental ICEalpha isoform. The deletion of 21 or 93 amino acids from the N-terminal propeptide in ICEbeta 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 ICEalpha, beta, 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 proICEalpha, beta, and , were stable for a prolonged period of time. Nevertheless, our in vitro translation reactions seemed to contain intermediate p34 species in proICEalpha and beta reactions, similar in size to proICE, but not in ICE reaction. This suggests that proICEalpha or beta 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)(2)/(p10)(2) 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)(2)/(ICE)(2) 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-1beta. Overexpression of pro-IL-1beta in Sf9 cells resulted in its cleavage to the 17.5-kDa active IL-1beta cytokine. (^2)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) .


FOOTNOTES

*
This work was supported by Research Grant AI 35035-01 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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].

§
To whom requests for reprints should be addressed: Dept. of Pharmacology, Thomas Jefferson University, Bluemle Life Sciences Bldg., 233 S. 10th St., Philadelphia, PA 19107. Tel.: 215-955-4634; Fax: 215-923-1098; litwack{at}calvin.jci.tju.edu.

(^1)
The abbreviations used are: ICE, interleukin-1-beta converting enzyme; RT-PCR, reverse transcription-polymerase chain reaction; AcNPV, Autographa californica nuclear polyhedrosis virus; Sf9, Spodoptera frugiperda cells; PAGE, polyacrylamide gel electrophoresis; IL, interleukin; bp, base pair(s); kb, kilobase pair(s).

(^2)
E. S. Alnemri, T. Fernandes-Alnemri, and G. Litwack, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank Dr. M. Summers for the baculovirus expression system and Dr. L. Wang for technical assistance.


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