©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Expression, Refolding, and Autocatalytic Proteolytic Processing of the Interleukin-1-converting Enzyme Precursor (*)

Paul Ramage (1)(§), Dominique Cheneval (2), Maria Chvei (1), Patrick Graff (1), Rene Hemmig (1), Richard Heng (2), Hans Peter Kocher (1), Andrew Mackenzie (2), Klaus Memmert (1), Laszlo Revesz (2), William Wishart (3)

From the (1) Departments of Biotechnology, (2) Bone and Joint, and (3) Signal Transduction, Sandoz Pharma Ltd., CH-4002 Basel, Switzerland

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The interleukin-1-converting enzyme is a heterodimeric cysteine protease that is produced as a 45-kDa precursor. The full-length precursor form of the enzyme was expressed in Escherichia coli as insoluble inclusion bodies. Following solubilization and refolding of the 45-kDa protein, autoproteolytic conversion to a heterodimeric form containing 10- and 20-kDa subunits was observed. This enzyme had catalytic activity against both natural (interleukin-1 precursor) and synthetic peptide substrates. The inclusion of a specific inhibitor (SDZ 223-941) of the converting enzyme in the refolding mixture prevented proteolytic processing to the 10-/20-kDa form. Similarly, refolding under nonreducing conditions also prevented processing. Time course experiments showed that the 10-kDa subunit was released from the 45-kDa precursor before the 20-kDa subunit, implying that the N-terminal portion of the precursor is released last and may play a regulatory role.


INTRODUCTION

Interleukin-1 (IL-1)() is a potent mediator of inflammation. IL-1 is produced in monocytes and monocytic cell lines as an inactive 31-kDa precursor (1, 2) . The active 17.5-kDa polypeptide is generated by specific proteolytic cleavage of the precursor molecule at Asp-Ala(3, 4) . It has been demonstrated that in human monocytes, proteolytic processing of the IL-1 precursor is carried out by a specific protease known as the IL-1-converting enzyme (ICE) (5, 6, 7, 8) . ICE has recently been purified, characterized, and cloned (8, 9, 10) and shown to be a heterodimeric cysteine protease (5, 9, 11) , comprising 10- (p10) and 20-kDa (p20) subunits in a 1:1 stoichiometric ratio. Structural data (12, 13) show that the active form of the enzyme is a tetramer formed from two of the p10/p20 heterodimers. The heterodimeric form is generated from a 45-kDa precursor (p45) by the proteolytic cleavage of four peptide bonds (Asp-Ser, Asp-Asn, Asp-Ser, and Asp-Ala; see Fig. 1) (8, 9) . ICE has an unusual specificity, requiring aspartic acid at the position N-terminal to the scissile peptide bond (P1) (9, 10, 14, 15) . This P1 specificity is shared with only two other enzymes from mammalian sources, granzyme B (16) , a serine protease from cytolytic lymphocytes, and prICE (17) , an enzyme that cleaves poly(ADP-ribose) polymerase. This very rare specificity suggests that a mechanism of autocatalytic processing is responsible for the generation of mature p10/p20 ICE. This theory is supported by several pieces of experimental evidence. The addition of purified ICE to the intact p45 precursor results in the generation of p10/p20 ICE (9) ; p45 expressed in the cytosolic fraction of SF9 cells using the baculovirus system is converted to the p10/p20 heterodimer form (18) , and a truncated form of the mouse ICE enzyme (minus amino acids 1-119) is expressed in Escherichia coli as a p10/p20 heterodimer (10) . Surprisingly, however, the p45 precursor, when expressed in rabbit reticulocyte lysate, remains stable (9) .


Figure 1: Primary structure of the human IL-1-converting enzyme precursor. Full-length p45 ICE may be cleaved at the four Asp- X cleavage sites indicated to produce mature heterodimeric p10/p20 ICE ( stippled regions). Cleavage at Asp-Serand Asp-Serresults in the formation of the polypeptides designated p24 and p14.



Here we show conclusively that following expression, purification, and refolding, the p45 precursor autocatalytically converts to the mature p10/p20 form of ICE.


EXPERIMENTAL PROCEDURES

Strains and Plasmid Construction The complete precursor ICE (p45) cDNA was cloned, via polymerase chain reaction amplification, from a commercially available human peripheral blood monocyte cDNA library (Clonetech 1050a), into vector pGI--PBR#13, under the control of the PL promoter and transfected into the E. coli strain SG936pCI857. The p45 cDNA clone was sequenced, and the DNA sequence was shown to agree with that reported by Thornberry et al. (9) . Fermentation of the Expression Strain The SG936pCI857 expression strain was grown in 1 liter of L-broth (NaCl, 10 g/liter; Difco Bacto-tryptone, 10 g/liter; BBL Bacto yeast extract, 5 g/liter), supplemented with ampicillin (0.1 g/liter), and kanamycin (0.04 g/liter) for 6 h at 30 °C. This preculture was used to inoculate 19 liters of fermentation medium ( L-broth with ampicillin, 0.1 g/liter) in a 30-liter steel fermenter. The bacteria were grown at 30 °C at an agitation speed of 200 rpm and an aeration rate of 1 (v/v/min). The expression of p45 ICE was induced by a temperature shift to 42 °C when an Avalue of 0.5-0.7 had been reached. The cells were harvested 4 h after induction; at which time, no further increase in Aabove a value of 3-4 was observed. The culture was harvested by centrifugation, and the cell paste was frozen until required. Inclusion Body Isolation and Purification Frozen E. coli wet cell pellets were suspended to 12.5% (w/v) in cell lysis buffer (50 m M Tris, pH 8.0; containing 2 m M DTT, 5 m M benzamidine-HCl, and 2 m M EDTA) and mixed by stirring for 1 h on ice. The cell suspension was lysed by passage through a Manton-Gaulin homogenizer (2 passes at 1200 bar) and then centrifuged for 30 min at 16,000 g. The resultant pellets were resuspended in lysis buffer and recentrifuged a further 3 times. Semipurified inclusion bodies were solubilized in 6 M guanidine-HCl, 25 m M DTT and purified by sequential preparative reversed phase HPLC on two Orpegen HD gel RP-7 s C8 (22 250 mm) columns using a water/acetonitrile buffer system. p45 containing fractions from the second Orpegen column were pooled and, following removal of organic solvent, lyophilized and stored at -20 °C. Refolding of p45 Lyophilized inclusion bodies were solubilized (1 mg/ml) in 50 m M Tris, pH 8.0 (containing 8 M urea, 50 m M DTT), for 1 h at room temperature. Following dilution with 3 volumes of 50 m M Tris, pH 8.0, 10 m M DTT, the refolding mixture was stirred overnight at room temperature and then dialyzed overnight at 4 °C against 2 80 volumes of 50 m M Tris, pH 8.0, containing 10 m M GSH. Refolding and p45 purification were monitored by analytical reversed phase HPLC using a 4.6 150-mm Polymer Laboratories PLRP-S 1000-Å column. Refolding in the Presence of a Specific ICE Inhibitor Refolding in the presence of SDZ 223-941, a P-site substrate based irreversible inhibitor, was carried out as described above except that at the dilution and dialysis steps inhibitor (SDZ 223-941 10 mg/ml stock in MeSO) was added to a 1.5 molar excess. Samples were removed at each step for SDS-PAGE and HPLC analyses. Gel Filtration Chromatography 4 ml of concentrated ICE was loaded onto a 1.6 60-cm column of Superdex 75 equilibrated in phosphate-buffered saline containing 10 m M DTT. 1-ml fractions were collected and analyzed on SDS-PAGE, and those found to contain p10/p20 ICE were pooled and tested. IL-1 Convertase Activity Determinations

Activity Determinations Using Recombinant hu-IL-1 Precursor as Substrate

ICE activity was detected by monitoring cleavage of recombinant IL-1 precursor to mature IL-1 using analytic HPLC. ICE samples were added (1:100 enzyme/substrate ratio) to 1 ml of recombinant hu-IL-1 precursor solution (330 µg/ml in phosphate-buffered saline, 10% glycerol) and incubated for 2 h at 37 °C. The incubation mixtures were then diluted 3-fold and analyzed on reversed phase HPLC (80 µl injection volume). Untreated precursor and recombinant hu-IL-1 were used as standards.

SDS-PAGE

SDS-PAGE was carried out under reducing conditions on 4-20% Novex gradient gels run according to the manufacturer's instructions.

N-terminal Sequence Analysis

N-terminal amino acid sequence determination by Edman degradation was performed on an Applied Biosystems 470A protein sequencer fitted with an HPLC on-line system 120A. Protein mixtures were run on 4-20% gradient gels (Novex) and electrophoretically transferred to polyvinylidine difluoride membranes (19) . Following identification of the protein bands, the blots were completely destained with 100% methanol, and the protein bands were cut out (1 10-mm pieces). These were then loaded into a continuous flow reactor (20) and microsequenced.


RESULTS

From 100 liters of fermentation broth, a 155-g wet cell pellet was obtained from which 22 g of inclusion bodies were isolated. At this stage, the purity of the p45 was no more than 25% (Fig. 2 a). Following solubilization and reversed-phase HPLC, the purity was increased to more than 80%, with p45 being observed essentially as a single band on SDS-PAGE (Fig. 2 a), which remained present following dilution to 2 M urea and the first dialysis. But during the second dialysis, this band gradually disappeared, being replaced by a large number of bands ranging in molecular mass from 36 to 8 kDa that had not been present in the starting material. Two of the most intense bands corresponded in molecular mass to the p10 and p20 subunits of ICE (Fig. 2 a). The gel was blotted onto polyvinylidine difluoride, and the most intense bands were sequenced. A total of seven bands with molecular masses of 45, 36, 27, 25, 23, 14, and 12 kDa, respectively, were cut and sequenced from the polyvinylidine difluoride membrane. The results (Figs. 3, a and b) show that all of the bands contained either the p45 N-terminal sequence or sequences generated following cleavage of an Asp- X bond. The bands at 25, 23, and 12 kDa were identified as p24, p20, and p10, respectively. The bands at 45 and 36 kDa contained only the p45 N-terminal sequence; the band at 36 kDa must, therefore, have been generated by cleavage at the C-terminal end of the protein. The band at 14 kDa consisted primarily (66%) of an elongated form of p10 containing the Ser-Asplinker (see Figs. 1 and 3 b). The band at 27 kDa contained 5 sequences; one identified as the p45 N-terminal (11%) and the remainder as fragments generated by cleavage after Asp(49%), Asp(15%), Asp(13%), and Asp(12%). When samples of the refolding mixture were removed at different times during the refolding and analyzed using SDS-PAGE, it was observed (Fig. 2 b) that the 36 and 14 kDa bands were generated first, followed by the bands at 27, 25, 23, and 12 kDa.


Figure 2: Recombinant human p45 ICE autocatalytically converts to p10/p20. Analysis of the autocatalytic conversion using reducing SDS-PAGE (Novex 4-20%) is shown. a, purification and autocatalytic cleavage. Lane 1, low molecular mass marker (Pharmacia Biotech Inc.); lane 2, p45 ICE inclusion body preparation; lane 3, p45 ICE purified from inclusion bodies by preparative reversed phase HPLC; lane 4, purified p45 ICE diluted down to 2 M urea prior to dialysis; lane 5, p45 ICE solution following dialysis at 4 °C against 2 80 volumes of dialysis buffer (50 m M Tris, pH 8.0; containing 10 m M GSH); lane 6, p10/p20 ICE purified on Superdex 75. b, the time dependence of autocatalytic conversion. Following dialysis against 2 100 volumes of dialysis buffer (in the absence of reducing agents), the retentate was concentrated 10-fold (2 m M oxidized glutathione was added to prevent autocatalysis during concentration); DTT was then added to 20 m M, and samples removed for SDS-PAGE at appropriate time intervals. Lane 1, molecular mass markers; lane 2, sample taken before dialysis; lane 3, sample taken after dialysis; lane 4, concentrated sample on addition of DTT; lane 5, sample 1 min after DTT addition; lane 6, sample 5 min after DTT addition; lane 7, sample 10 min after DTT addition; lane 8, sample 15 min after DTT addition; lane 9, sample 30 min after DTT addition; lane 10, sample 45 min after DTT addition. c, the influence of different reducing agents on the rate of autocatalysis and the effect of a specific ICE inhibitor on autocatalysis. Lane 1, molecular mass markers; lane 2, p45 ICE solution (200 µg/ml p45 in 2 M urea, 50 m M Tris, 10 m M DTT, pH 8.0) prior to dialysis; lane 3, p45 ICE solution dialyzed against 50 m M Tris, 10 m M GSH, pH 8.0; lane 4, p45 ICE solution dialyzed against 50 m M Tris, 10 m M DTT, pH 8.0; lane 5, p45 ICE solution dialyzed against 50 m M Tris, 10 m M -mercaptoethanol, pH 8.0; lane 6, p45 ICE solution dialyzed against 50 m M Tris, pH 8.0, containing no reducing agents; lane 7, p45 ICE solution (containing a 1.5 molar excess of the specific ICE inhibitor SDZ 223-941) following dialysis against 50 m M Tris, pH 8.0 (containing 10 m M GSH and a 1.5 molar excess of 223-941); lane 8, p45 ICE solution dialyzed against 50 m M Tris, pH 8.0 (no reducing agents) and then made 10 m M with respect to GSH and incubated for 2 h at room temperature.



When analyzed on SDS-PAGE, refolding mixtures containing the specific ICE inhibitor SDZ 223-941 showed a greatly reduced generation of lower molecular weight species, in contrast to what was observed in reactions containing only GSH, DTT, or -mercaptoethanol (Fig. 2 c). Similarly such refolding mixtures were shown by analytical HPLC to be inactive using recombinant IL-1 precursor as substrate (Fig. 4 d). When the dialysis step of refolding was carried out under nonreducing conditions by omission of the thiol reagents, p45 remained similarly undegraded. Subsequent addition of GSH to 10 m M resulted in rapid breakdown of p45 (Fig. 2 c). When GSH in the dialysis buffer was replaced by either -mercaptoethanol or DTT at the same concentration, a similar generation of lower molecular weight fragments was observed (Fig. 2 c). However, dialysis with DTT led to a further degradation of p10 not seen with either GSH or -mercaptoethanol. Incubation of recombinant hu-IL-1 precursor with p45 solution after dialysis was shown by HPLC (Fig. 4 c) to result in an almost complete breakdown of IL-1 precursor yielding mature IL-1 and three precursor fragments. Determination of the ICE activity of this preparation using a synthetic peptide substrate revealed a specific activity of 0.369 µmol/mg/min (not shown). When a concentrated solution of p45 dialyzed under reducing conditions was run on Superdex 75, a peak was eluted with an apparent molecular mass of 34,500 daltons that was shown by SDS-PAGE (Fig. 2 a) to contain virtually pure p10/p20. When tested against recombinant IL-1 precursor, this material was shown to possess ICE activity (Fig. 4 e).


Figure 4: Specific ICE activity of autocatalytically processed p45. Analytical reversed phase HPLC of different p45 ICE fractions incubated at 37 °C for 2 h with a 100-fold excess of recombinant hu-IL-1 precursor (500 µg/ml in phosphate-buffered saline, 10% (v/v) glycerol) and then diluted 3-fold with HPLC buffer A. The positions of IL-1, the IL-1 precursor, and fragments derived following cleavage of the IL-1 precursor are indicated. a, control profile, r-hu-IL-1 precursor; b, control profile, r-hu-IL-1; c, specific ICE activity of p45 ICE dialyzed against 50 m M Tris, 10 m M GSH, pH 8.0. d, specific ICE activity of p45 ICE diluted and dialyzed in the presence of a 1.5 molar excess of the ICE inhibitor SDZ 223-941. e, specific ICE activity of autocatalytically derived p10/p20 ICE purified by chromatography on Superdex 75.




DISCUSSION

We have demonstrated that, following refolding by dilution and subsequent dialysis, purified recombinant p45 ICE is converted to p10/p20 ICE. Conversion occurs in a time-dependent manner through a series of intermediates. Identification of these through sequence analysis has given us an insight into the mechanism of conversion. The process begins slowly during the second dialysis step as the chaotroph concentration is reduced and three prominent products are seen. These are a 36-kDa C-terminally truncated p45, p10, and p10 carrying the Ser-Asplinker. At this stage, little or no p20 or p24 are observed. This suggests that the first step in the conversion is the removal of p10 plus linker by cleavage at Aspfollowed rapidly or simultaneously by the removal of the linker by cleavage at Asp. The p36 band, which contains only the p45 N-terminal sequence, must therefore represent p45 minus Ser-Hisand is itself further processed down to p20 (presumably via p24, as the intensities of the p24 and p20 bands do not increase significantly until p14 and p10 have already been formed and p36 starts to decrease in intensity; Fig. 2 b). Therefore, the p10/p20 form is clearly formed via processing through several intermediates, each itself being formed by cleavage at Asp- X (Fig. 5). Conversion to p10/p20 was abolished by the inclusion of SDZ 223-941, a P-site substrate-based irreversible inhibitor of ICE. The end product of the conversion was an ICE preparation of far greater stability and higher specific activity than ICE produced by the co-refolding of separately expressed p10/p20 subunits.() Gel-filtration chromatography resulted in preparations containing virtually pure active ICE with an apparent molecular mass of 34,500 Da, similar to the previously reported figure of 29,000 Da (13) . Lower molecular mass protein products caused by further autodegradation upon concentration of ICE (12, 13) were effectively removed.


Figure 5: The autocatalytic processing of p45 to p10/p20 ICE occurs in an ordered manner through several intermediates. Sequential positions of cleavage are marked with an arrow. The stippled and shaded regions represent the polypeptides removed by autocatalytic cleavage. a, intact p45 ICE; b, cleavage at Asp-Ser, resulting in the formation of p36 and p14; c, cleavage at Asp-Alain p14, releasing p10; d, cleavage at Asp-Serin p36, releasing p24; e, cleavage at Asp-Asnin p24, releasing p20.



Until now, the evidence for the autocatalytic conversion of p45 ICE to p10/p20 ICE has been strong but circumstantial (8, 9, 10, 11) . The expression of p45 in the cytosolic fraction of SF9 cells and the subsequent detection of a number of lower molecular weight components including p10/p20 ICE (18) , could conceivably have been due to the proteolytic ``activation'' of p45 by SF9 proteases. Earlier work (9) had shown that p45 produced in a rabbit reticulocyte lysate system remained in the precursor form until mature ICE had been added, leading the authors to believe that another protease may have been required to activate p45. The expression of a truncated form (minus amino acids 1-119) of murine p45 in E. coli resulted in the production of p10/p20 (10) , suggesting that the N-terminal 119 amino acids of p45 may play a regulatory role in autocatalytic conversion.

The reaction that we have observed has been carried out in vitro using p45 expressed as insoluble inclusion bodies in E. coli. These inclusion bodies were purified under strongly denaturing conditions prior to refolding and would therefore be unlikely to have contained active proteases of bacterial origin. In a control experiment (not shown) an inclusion body fraction from the same host cell line, but expressing an unrelated protein was enriched using HPLC and a fraction corresponding to the position of elution of p45ICE collected and refolded using identical conditions to those used for p45 ICE. No protease activity was detected using the IL-1 precursor and synthetic peptides as substrates (data not shown). The cleavage products that we observed were those produced by cleavage at the known cleavage sites in p45 (8, 9) . Only the faint band at 27 kDa contained sequences that were produced by cleavage at other Asp- X sites. It has been reported that although the protein prICE recognizes the same tetrapeptide cleavage site as ICE, prICE does not cleave the IL-1 precursor, nor does ICE have any activity against the prICE substrate, poly(ADP-ribose) polymerase (17) . It is extremely unlikely that a contaminating protease with Asp- X specificity could have been responsible for the specific cleavage observed and that this cleavage was therefore autocatalytic.

Following dilution under reducing conditions, dialysis carried out in the absence of GSH, DTT, or -mercaptoethanol prevented autocatalytic processing. The subsequent addition of GSH resulted in rapid autocatalysis, showing clearly that reducing agents play a key role in triggering the autocatalytic conversion of p45 to ICE.

When p45 was observed to be expressed in an intact form in a rabbit reticulocyte lysate system (9) , this stability may have been due to the relatively low thiol content of the translation mixture. How ICE autocatalysis is controlled in vivo remains to be seen. However, the fact that an N-terminally truncated form (minus amino acids 1-119) of the mouse enzyme expressed in E. coli was produced as a p10/p20 heterodimer indicates a role for amino acids 1-119 in the regulation of autocatalysis. We have shown that removal of amino acids 1-119 is not necessary for autocatalysis to occur and that the regulatory mechanism can, in-vitro, be bypassed by high concentrations of low molecular weight thiols. The strong effects exerted by thiols suggest that the three closely located cysteines at positions Cys, Cys, and Cyscould play a role in the regulation of enzyme activity. On the other hand, the dependence of ICE autocatalysis on low molecular weight thiols might merely be a reflection of the fact that mature p10/p20 ICE requires DTT concentrations in the order of 10 m M for reactivation (21) .

We have shown that the interleukin-1-converting enzyme is produced by an autocatalytic conversion of the 45-kDa precursor in vitro. The mechanism controlling autocatalytic conversion remains unclear, but our results show that low molecular weight thiols such as glutathione play a key role in triggering autocatalytic conversion in vitro.


FOOTNOTES

*
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.

§
To whom correspondence should be addressed. Tel: 41-61-324-7230; Fax: 41-61-324-6303.

The abbreviations used are: IL-1, interleukin-1; hu-IL-1, human IL-1; ICE, IL-1-converting enzyme; DTT, 1,4-dithio- DL-threitol; HPLC, high performance liquid chromatography; PAGE, polyacrylamide gel electrophoresis.

P. Ramage and A. Mackenzie, unpublished results.


REFERENCES
  1. Mosley, B., Urdal, D. L., Prickett, K. S., Larsen, A., Cosman, D., Conlon, P. J., Gillis, S., and Dower, S. K. (1987) J. Biol. Chem. 262, 2941-2944 [Abstract/Free Full Text]
  2. Fuhlbrigge, R. C., Fine, S. M., Unanue, E. R., and Chaplin, D. D. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 5649-5653 [Abstract]
  3. Black, R. A., Kronheim, S. R., Cantrell, M., Deeley, M. C., March, C. J., Prickett, K. S., Wignall, J., Conlon, P. J., Cosman, D., Hopp, T. P., and Mochizuki, D. Y. (1988) J. Biol. Chem. 263, 9437-9442 [Abstract/Free Full Text]
  4. Hazuda, D., Webb, R. L., Simon, P., and Young, P. (1989) J. Biol. Chem. 264, 1689-1693 [Abstract/Free Full Text]
  5. Black, R. A., Kronheim, S. R., and Sleath, P. R. (1989) FEBS Lett. 247, 386-390 [CrossRef][Medline] [Order article via Infotrieve]
  6. Kostura, M. J., Tocci, M. J., Limjuco, G., Chin, J., Cameron, P., Hillman, A. G., Chartrain, N. A., and Schmidt, J. A. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 5227-5231 [Abstract]
  7. Kronheim, S. R., Mumma, A., Greenstreet, T., Glackin, P. J., Van Ness, K., March, C. J., and Black, R. A. (1992) Arch. Biochem. Biophys. 296, 698-703 [Medline] [Order article via Infotrieve]
  8. Miller, D. K., Ayala, J. M., Egger, L. A., Raju, S. M., Yamin, T.-T., Ding, G. J.-F., Gaffney, E. P., Howard, A. D., Palyha, O. C., Rolando, A. M., Salley, J. P., Thornberry, N. A., Weidner, J. R., Williams, J. H., Chapman, K. T., Jackson, J., Kostura, M. J., Limjuco, G., Molineaux, S. M., Mumford, R. A., and Calaycay, J. R. (1993) J. Biol. Chem. 268, 18062-18069 [Abstract/Free Full Text]
  9. Thornberry, N. A., Bull, H. G., Calaycay, J. R., Chapman, K. T., Howard, A. D., Kostura, M. J., Miller, D. K., Molineaux, S. M., Weidner, J. R., Aunins, J., Elliston, K. O., Ayala, J. M., Casano, F. J., Chin, J., Ding, G. J.-F., Egger, L. A., Gaffney, E. P., Limjuco, G., Palyha, O. C., Raju, S. M., Rolando, A. M., Salley, J. P., Yamin, T.-T., Lee, T. D., Shively, J. E., MacCross, M., Mumford, R. A., Schmidt, J. A., and Tocci, M. J. (1992) Nature 356, 768-774 [CrossRef][Medline] [Order article via Infotrieve]
  10. Molineaux, S. M., Casano, F. J., Rolando, A. M., Peterson, E. P., Limjuco, G., Chin, J., Griffin, P. R., Calaycay, J. R., Ding, G. J.-F., Yamin, T.-T., Palyha, O. C., Luell, S., Fletcher, D., Miller, D. K., Howard, A. D., Thornberry, N. A., and Kostura, M. J. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 1809-1813 [Abstract]
  11. Cerretti, D. P., Kozlosky, C. J., Mosley, B., Nelson, N., Van Ness, K., Greenstreet, T. A., March, C. J., Kronheim, S. R., Druck, T., Cannizzaro, L. A., Huebner, K., and Black, R. A. (1992) Science 256, 97-100 [Medline] [Order article via Infotrieve]
  12. Wilson, K. P., Black, J. F., Thomson, J. A., Kim, E. E., Griffith, J. P., Navia, M. A., Murcko, M. A., Chambers, S. P., Aldape, R. A., Raybuck, S. A., and Livingston, D. J. (1994) Nature 370, 270-275 [CrossRef][Medline] [Order article via Infotrieve]
  13. Walker, N. P. C., Talanian, R. V., Brady, K. D., Dang, L. C., Bump, N. J., Ferenz, C. R., Franklin, S., Ghayur, T., Hackett, M. C., Hammill, L. D., Herzog, L., Hugunin, M., Houy, W., Mankovich, J. A., McGuiness, L., Orlewicz, E., Paskind, M., Pratt, C. A., Reis, P., Summani, A., Terranova, M., Welch, J. P., Xiong, L., Moller, A., Tracey, D. E., Kamen, R., and Wong, W. W. (1994) Cell 78, 343-352 [Medline] [Order article via Infotrieve]
  14. Sleath, P. R., Hendrickson, R. C., Kronheim, S. R., March, C. J., and Black, R. A. (1990) J. Biol. Chem. 265, 14526-14528 [Abstract/Free Full Text]
  15. Howard, A. D., Kostura, M. J., Thornberry, N., Ding, G. J. F., Limjuco, G., Weidner, J., Salley, J. P., Hogquist, K. A., Chaplin, D. D., Mumford, R. A., Schmidt, J. A., and Tocci, M. J. (1991) J. Immunol. 147, 2964-2969 [Abstract/Free Full Text]
  16. Caputo, A., James, M. N. G., Powers, J. C., Hudig, D., and Bleakley, R. C. (1994) Struct. Biol. 1, 364-367
  17. Lazebnik, Y. E., Kaufmann, S. H., Desnoyers, S., Poirier, G. G., and Earnshaw, W. C. (1994) Nature 371, 346-347 [CrossRef][Medline] [Order article via Infotrieve]
  18. Wang, X.-M., Helaszek, C. T., Winter, L. A., Lirette, R. P., Dixon, D. C., Ciccarelli, R. B., Kelley, M. M., Malinowski, J. J., Simmons, S. J., Huston, E. E., Koehn, J. A., Kratz, D., Bruckner, R. C., Graybill, T., Ator, M. A., Lehr, R. V., and Stevis, P. E. (1994) Gene ( Amst.) 145, 273-277 [Medline] [Order article via Infotrieve]
  19. Matsudaira, P. (1987) J. Biol. Chem. 262, 10035-10038 [Abstract/Free Full Text]
  20. Shively, J. E., Miller, P., and Ronk, M. (1987) Anal. Biochem. 163, 517-529 [Medline] [Order article via Infotrieve]
  21. Thornberry, N. A., Peterson, E. P., Zhao, J. J., Howard, A. D., Griffin, P. R., and Chapman, K. T. (1994) Biochemistry 33, 3934-3940 [Medline] [Order article via Infotrieve]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.