[CANCERRESEARCH55. 6045-6052. December 15. 19951

Advances in Brief

Mch3, a Novel Human Apoptotic Cysteine Protease Highly Related to CPP321

Teresa Fernandes-Alnemri, Atsushi Takahashi, Robert Armstrong, Joseph Krebs, Lawrence Fritz, Kevin J. Tomaselli,Ljjuan Wang, Zailin Yu, Carlo M. Croce, Guy Salveson, William C. Eamshaw, Gerald Litwack, and Emad S. Alnemri2

Departments of Pharmacology IT. F-A., L W., G. L, E. S. A.] and Microbiology and Immunology (1 Y.. C. M. C.]. Jefferson Cancer Institute, Jefferson Medical College,Philadelphia. Pennsylvania 19107; Department of Cell Biology and Anatomy. Johns Hopkins School of Medicine, Baltimore, Maryland 21205 [A. T., W. C. El: Cell BiologySection, IDUN Pharmaceuticals, San Diego, California 92121 (R. A.. J. K.. L F.. K. J. Ti; and Department of Pathology, Duke University Medical School, Durham,North Carolina 277/0 (G. 5.1

Abstract

Recent evidence suggests that mammalian cysteine pretenses related toCaenorhabditis elegans CED-3 are key components of mammalian pro

grammed cell death or apoptosis. We have shown recently that the CPP32and Mch2a cystelne proteases cleave the apoptotic markers poly(ADP.ribose) polymerase (PARP) and lamins, respectively. Here we report thecloning of a new Ced-3/interleukin 1@-convertlng enzyme-related gene,

designated Mch3, that encodes a protein with the highest degree of ho

mology to CPP32 compared to other family members. An alternativelyspliced isoform, named Mch3@, was also identifIed, Bacterially expressedrecombinant Mch3 has intrinsic autocatalytic/autoactivationactivity. Thespecific activity of Mch3a toward the peptide substrate DEVD-7-amlno4-methylcoumarinand PARP resemblesthat of CPP32. Like interleukin1@3-converting enzyme and CPP32, the active Mch3a is made of two

subunits derived from a precursor (proMch3a). It was of interest thatrecombinant CPP32-p17 subunit can form an active heteromeric enzymecomplex with recombinant Mch3a-p12 subunit and vice versa, as determined by the ability of the heteromeric complexes to induce apoptosis in

Sf9 cells. These data suggest that proMch3a and proCPP32 can interactto form an active Mch3a/CPP32 heteromeric complex. We also provideevidence that CPP32 can efficiently cleave proMch3a, but not the opposite, suggesting that Mch3a activation in vivo may depend in part onCPP32 activity. The high degree of conservation in structure and specific

activity and the coexistence of Mch3a and CPP32 in the same cell suggests

that the PARP cleavage activity observed during apoptosis cannot solelybe attributed to CPP32 but could also be an activity of Mch3a.

Introduction

The growing ICE3/Ced-3 family of cysteine proteases includemammalian ICE (1, 2), NEDD2 (ICH-1; Refs. 3 and 4), CPP32 (5),Mch2 (6), TX (ICH-2, ICE@J 1!; Refs. 7—9)and ICE@1—III(9)proteins as well as the C. elegans Ced-3 cell death protein (10). Theseproteases fall into two subfamilies composed of ICE, NEDD2, TX,and ICE@1—IIIon the one hand and CED-3, CPP32, and Mch2 on theother. Common features of this family include conservation of theactive site QACRO pentapeptide, ability to induce apoptosis whenoverexpressed in a heterologous system, and requirement for proteolytic cleavage of the proenzymes at conserved aspartate cleavage sitesfor activation. Our laboratory and others have shown recently that

Received 9/29/95; accepted 10/27/95.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

I This work was supported in part by a grant from [DUN Pharmaceuticals to (E. S. A.),

by Grant Al 35035-01 from NIH (to G. L.), and by Grant CA69008 from NIH(to W. C. E.).

2 To whom requests for reprints should be addressed, at Department of Pharmacology,

Jefferson Medical College, Bluemle Life Sciences Building, 233 South 10th Street,Philadelphia, PA 19107. Phone: (215) 955-4632; Fax: (215) 923-1098; E-mail:E.Alnemri@Iac.jci.tju.edu.

3 The abbreviations used are: ICE, interleukin 1(3-converting enzyme; Mch3, roam

malian Ced-3 homologue 3; PARP, poly(ADP-ribose) polymerase; IPTG, isopropyl-lthio-@-o-galactopyranoside; GST, glutathione S-transferase; AMC, 7-amino-4-methylcoumarin; AcNPV, Autographa californica nuclear polyhedrosis virus; SI'), Spodopterafrugiperda cells: kDa, kilodalton.

human CPP32 can cleave PARP, an event that is diagnostic ofapoptotic death in many cell types (6, 11—13).This activity, its highhomology to Ced-3, and the ability of a tetrapeptide aldehyde contaming the P1—P4amino acid sequence of the PARP cleavage site toinhibit PARP cleavage and the apoptotic events in vitro, led to thesuggestion that CPP32 is the human equivalent of Ced-3 (12). However, new evidence suggests that CPP32 might not be the only humanequivalent of Ced-3. First, Mch2 has about the same homology toCed-3 as does CPP32, and it can cleave both PARP and lamins,yielding cleavage products indistinguishable from those producedduring apoptosis (6). Second, PARP cleavage activity is not restrictedto CPP32 and Mch2 but can also be observed with ICE, TX, andNEDD2, albeit at a higher protease concentration (14). Finally, wereport here the cloning, expression, and characterization of a novelcysteine protease highly related to CPP32. The new protease, namedMch3,4 can cleave PARP, and its activity is indistinguishable fromthat of CPP32. In addition, recombinant p20 or p12 subunits derivedfrom Mch3 can form active heteromeric complexes with recombinantp17 or p12 subunits derived from CPP32. Like ICE, CPP32, andMch2 (6), bacterially expressed recombinant Mch3 has intrinsic autocatalytic/autoactivation activity.

Materials and Methods

Cloning of Human Mch3. A l0-,&laliquot of human Jurkat A Uni-ZAPXR cDNA library (5) containing @@@l08plaque-forming units was denatured at99°Cfor 5 mm and used as a template for PCR amplification with a degenerateprimer encoding the pentapeptide GSWFI/GSWYI and T3 vector-specificprimer (Stratagene) as described previously (6). A l0-@.daliquot of the primaryamplification product was then used as a template for a secondary PCR

amplification with primer T50-prl (CCGTGGAATAGGCGAAGAG)derivedfrom GenBank sequence T50828 and a second vector-specific primer SK-Zap(CAGGAATFCGGCACGAG) located downstream of the T3 primer. Thesecondary amplification products were cloned into a SmaI cut pBluescript IIKS@vector. All clones were screened by PCR using the QACRO degenerateprimer and SK-Zap primer. Clones that were positive for the presence of theQACROcodingsequencewerethen subjectedto DNAsequencingusingtheT3 and Ti sequencing primers (Stratagene). This resulted in identification ofa Ced3IICE-like partial cDNA with high homology to CPP32. The partialcDNA was then excised from the vector, radiolabeled, and used to screen theoriginal Jurkat AUni-ZAP XR cDNA library. Positive Aclones were purified,rescued into the pBluescript II SK plasmid vector, and sequenced.

Construction of Transfer Vectors and Recombinant Baculoviruses.

The Mch3 cDNA was amplified by PCR using primers T50-pr3 (GCCATAAACTCTTCCTCACTr) and T50-pr4 (ATGGCAGATGATCAGGGC) and

subcloned into the pBluescript II SK vector. The Mch3 sequence was then

excised with BamHl and subcloned into a BamHl cut pVLl393 to generatepVL-Mch3cttransfer vector. The cDNAs encoding the p20 and p12 subunits ofMch3a were amplified by PCR using the following primers: p20 subunit,T50-pr4 and Mch3-p20-tag (CTAGTCGGCCTGGATGCCATC);p12 subunit,

4 The sequences reported in this paper have been deposited in the GenBank data base

(accession numbers u37448 and u37449).

6045

Research. on December 26, 2020. © 1995 American Association for Cancercancerres.aacrjournals.org Downloaded from

@17PSLFSKKKKNVTMRSIKTTRDRVPTYQYNMNFEKLGKCI

ITAcCGTCCCCTrCATAAGAAGAA@AAAATGTCACC.@TGcGATCCATcAAGACcACCCGGGACCGAGTGCCTACATATCAGTACAACATGAATTTTGAAAAGCTGGGCAAATGCATCA

INNKNFDKVTGMGVRNGTDKDAEALFKCFRSLGFDVIVYNTAATAAACMCAAGAACTrTG@TAAAGTGACAGGTATt@GcGTTCGAAAcGGAACAGACAAAG@TGCcGAGGCGCTCTTCAAC,TGCTTCCGAAGCCTGGGT1'rTGACGTGATTGTCTATA

DCSCAKMQDLLKKASEEDHTNAACFACILLSHGEENVIYGATGACTGCTTGTGCCAAG@TGCAAGATCTGCTTAAAAAGCTTCTGAAGAGGAcCATACAAATGCcGcCTGCI'TCGcCTGCATCCTCTTAAGCCATGGAGAAGAAAATGTAATrTATG

ATGGMTCTTMESCK

D G V T P I K D L T A H F R G D R C K T L L E K P K L F F IlQ A C R GIT E L D

IISVTQAGVQRRDLGRLQPPPP

pj2@DGIQADSGPINDTDANPRYKIPVEADFLFAYSTVPGYYSW

MMASRPTRGPSMTQMLILDTRSQWKLTSSSPIPRFQAITRRSPGRGSWFVQALCSILEE}IGKDLEIMQILTRVNDRVARH

GGAQEEAPGLCKPSAPSWRSTEKTWKSCRSSPG*FESQSDDPHFHEKKQIPCVVSMLTKELYFSQ*

CLONING OF Mch3

Mch3aMch3@

. G@GAGACTGTGCCAGTC17

p20 ____@@@

MADDQGCIEEQGVEDSANEDSVDAKPDRSSFV 32137

72257

112377

152497

152

192617

181

232737221

272857253

GG@CCGAAG@GGATChGCCTTGTGG

30397710971217133714571577169718171937205721772297

Fig. I . Nucleotide and predicted amino acid sequence of the human Mch3a and Mch3@3.Colinear alignment of Mch3a and Mch3(3 cDNAs. The nucleotide sequence of Mch3@ thatis different from that of Mch3a is shown below the nucleotide sequence of Mch3a. The predicted amino acid sequence of Mch3a is shown above the nucleotide sequence. The predictedamino acid sequence of Mch33 that is different from that of Mch3a is shown below the nucleotide sequence spliced sequences in Mch3a and @3.The underlined Mch3a sequenceis deleted in Mch3@ and is replaced by the intronic sequence shown below it. The putative active site pentapeptide of Mch3a is boxed. Horizontal arrows, putative p20, p17, and p12.Vertical arrow, intron location. Amino acid and nucleotide residues are numbered to the right of each sequence.

ATGTGGTTGC'I'T 2309

Mch3-pl2-atg (ATGTCGGQGCCCATCAATGAC) and T50-pr9 (GACCCATTGCTI'CTCAGC). The PCR products were then cloned into a SmaI cutpVLl393 to generate pVL-Mch3-p20 and pVL-Mch3-p12 transfer vectors.The recombinant transfer vectors were then used to generate recombinantbaculoviruses.

Northern Blot Analysis. Tissue distribution analysis of Mch3 mRNA wasperformed on Northern blots prepared by Clontech containing 2 @xg/laneofpoly(Ai RNA. A radioactive Mch3a riboprobe was prepared using a Sow!linearized pBluescript II SK-Mch3a as a template for Ti RNA polymerase inthe presence of [&32P]ATP. The blots were hybridized, washed, and thenvisualized by autoradiography.

Expression of Mch3 in Bacteria and Assay of Enzyme Activity. TheGST-Mch3al and GST-Mch3a2 cDNAs were subcloned in-frame into theBamHI site of the bacterial expression vector pGEX-2T (Pharmacia Biotech,

Inc.). The GST-Mch3a3 cDNA was subcloned in-frame into the BamHI/EcoRIsite of pGEX-2T. Bacterial extracts were prepared and assayed as described

recently (6). YVAD-CHO and -AMC were purchased from Bachem Bioscience (King of Prussia, PA). Recombinant His-tagged Crm A was pun

fled as described (1 1). Synthesis of DEVD-CHO and -AMC will bedescribedelsewhere.5

In Vitro Transcription and Translation. Mch3a, Mch3fJ,GST-Mch3a,and GST-CPP32 cDNAs were subcloned into the pBluescript II KS@plasmid

under the T7 promoter. The vectors were linearized with the appropriaterestriction enzyme and used as templates for 11 RNA polymerase. The in vitrosynthesized mRNAs were then used for in vitro translation with reticulocyte

lysates as described previously (15).

Results and Discussion

Cloning of Mch3. An approach combining information from theGenBank database of human expressed sequence tags and PCR wasused to identify and clone a novel member of the family of Ced-3/ICE-like apoptotic cysteine proteases from the human Jurkat T-cellline. Human Ced-3IICE-like sequences present in a unidirectionalJurkat cDNA library were first enriched using degenerate PCR primers encoding the conserved GSWFIJGSWYI pentapeptides (6). Theenriched library was amplified with a primer derived from an expressed sequence tag (T50828), and the resulting partial cDNA wassequenced to confirm its identity and then used as a probe to obtainthe full-length cDNA as described in “Materialsand Methods.―Several cDNA clones were isolated. One cDNA, named Mch3a, containsan open reading frame of 909 bp that encodes a 303-amino acidprotein with a predicted molecular mass of —34kDa (Fig. 1). Theinitiator methionine at nucleotide 44 conforms to the consensus Kozaktranslation initiation sequence (16). A second cDNA clone, named5 K. J. Tomaselli, manuscript in preparation.

6046

Research. on December 26, 2020. © 1995 American Association for Cancercancerres.aacrjournals.org Downloaded from

I MCh3U@ I

I

I____Mch3B @@%@%4

CLONING OF Mch3

Mch3U

ExonA

Mch3@

@ecAGFig. 2. Alternative splicing of Mch3 mRNA. A

diagram of alternative transcripts of Mch3 and theirpredicted protein products. Boxes. exons identifiedas exon A and exon B. The solid line between exonA and B indicates intron A, which is 767 bp. •and

@ in Mch3f3 protein. amino acids encoded by intronic and frame shifted sequences. Inset. in vitrotranslated and 35S-labeled Mch3a and f3 proteinsanalyzed on a 10—20%gradient SDS-polyacrylamide gel and visualized by autoradiography. Stop

Stop

Mch3@, was also identified and found to contain a deletion andinsertion corresponding to nucleotides 488—592 of the Mch3a sequence (amino acids 149—183;Fig. I). Mch3f3 also has a longer5 ‘-nontranslatedsequence. Exon/intron analysis of the Mch3 genomicregion that corresponds to the deletion/insertion in Mch3f3 revealedthat the Mch3@ mRNA resulted from two simultaneous alternativesplicing events involving an exon and an intron shown as exon A andintron A in Fig. 2. The first event caused the deletion of nucleotides488—592 of the Mch3cs sequence due to the use of an alternativesplice donor located within the coding region of exon A and analternative splice acceptor located within intron A (Fig. 2). Thesecond splicing event caused an insertion of a 74-bp intronic sequencedue to the use of an alternative splice donor located within intron Aand the normal splice acceptor of intron A. All the alternative splicedonor/acceptor sites used in these events conform to the GT/AG rule(Fig. 2). As a result of the deletion and insertion, Mch3f3 cDNA didnot maintain the same reading frame as Mch3a after amino acid 148.The new reading frame in Mch3@3does not encode a QACRG pentapeptide sequence, and it terminates with a TGA stop codon corresponding to bp 837—839of Mch3a (Fig. 1). Mch3@3encodes a proteinof 253 amino acids with a predicted molecular mass of —28kDa. Invitro translated Mch3a and Mch3j3 migrate as 36- and 33-kDa protein

products, respectively (Fig. 2). The smaller translation products seenin the Mch3a and Mch33 translation reactions are probably internallytranslated products. We do not yet know the function of Mch3@.However, similar to the alternatively spliced Ich-l isoform (Ich-ls;Ref. 4), Mch3@ could be a negative regulator of apoptosis and couldinhibit the activity of the parental enzyme by acting as a dominantinhibitor.

Mch3 Is a Cysteine Protease Highly Related to CPP32. Thepredicted full-length Mch3a protein sequence shows the highest homology to human CPP32 (5). Mch3a shares an overall —53%identity(67%similarity)with CPP32(Fig. 3), comparedto —37%identity(55% similarity) with Mch2a (6) and —33%identity (55% similarity)

with CED-3 (10). Mch3a shows less than 30% identity with otherfamily members such as ICE, NEDDIICH-l, TX (ICH-2, ICE@111;Refs. 7—9),or ICE@, 111 (9). In addition to the conservation of theactive site QACRG pentapeptide, the predicted structure of Mch3aappears to be similar to CPP32 (5, 12). CPP32 is cleaved at Asp28 andAsp175 to generate two polypeptides with molecular masses of 17(pl'7) and 12 (pl2) kDa that form the active CPP32 enzyme complex(12).Onthebasisof thehighhomologybetweenMch3aandCPP32,the most probable cleavage sites in Mch3a are Asp53 and Aspl98(Figs. 1 and 3). Cleavage at these sites would generate two polypeptides equivalent to the p17 and p12 subunits of CPP32. However,there are three potential aspartic acid cleavage sites at positions 15,20, and 23 that could be used to remove a short propeptide duringprocessing of Mch3a to the active enzyme. In fact, the tetrapeptideDSVD (amino acids 20—23of Mch3a) is very similar to the DEVDtetrapeptide substrate of CPP32 (6, 12). This suggests that Mch3cxmight be a substrate for CPP32, as we have described below. Inaddition, three Asp cleavage sites (Aspl93, Asp204, and Asp206)located between the two subunits may serve as potential processingsites to separate the two subunits.

Tissue Distribution of Mch3. The tissue distribution of Mch3 wasanalyzed by Northern blot analysis of poly(A@) RNA isolated fromdifferent human tissues. As shown in Fig. 4, a major 2.4-kb Mch3message is detectable in all tissues examined. The lowest expressionof Mch3 mRNA is seen in whole brain. Examination of Mch3 mRNAin different regions of the brain also shows low but detectable expression. A similar tissue distribution was also seen with CPP32 mRNA,although the CPP32 mRNA is more abundant than Mch3 mRNA inbrain tissues (data not shown). The size of the Mch3 mRNA isconsistent with the length of the cloned Mch3a and @3cDNAs (Fig. 1).Two less abundant messages of (0.8 and 3.3 kb) are also detectable insome tissues such as the small intestine. The larger message could bean incompletely processed Mch3 mRNA or an alternatively spliced

6047

Mch3a mRNA

KDa

-94-67

Mch3@mRNA

Research. on December 26, 2020. © 1995 American Association for Cancercancerres.aacrjournals.org Downloaded from

CLONING OF Mch3

Mch3a

@PP32

1

1

51

36

101

78

151

128

201

178

251

225

301

275

MADDQGCIEEQGVEDSANEDSVDAKPDRSSFVPSLFSKKKKNVTMRS IKT

...I:III.I.:.:I.:MENTENSVDSKS 1K .NLEPKI IHGSESMDSGI SLDN

TRDRVPTYQYNMNFEKLGKCI I INNKNFDKVTGMGVRNGTDKDAEALFKC

111111111.1 III. 1.111 II..IS YKMDYPEMGLCIIINNKNFHKSTGMTSRSGTDVDAANLRET

FRSLGFDVIVYNDCSCAKMQDLLKKASEEDHTNAACFACILLSHGEENVIII.I::(FRNLKYEVRNKNDLTREEIVELMRDVSKEDHSKRSSFVCVLLSHGEEGI I

YGKDGVTP IKDLTAHFRGDRCKTLLEKPKLFF IQACRGTELDDGIQADSG

FGTNGPVDLKKITNFFRGDRCRSLTGKPKLFI IQACRGTELDCGIETDSG

P INDTDANPRYKIPVEADFLFAYSTVPGYYSWRSPGRGSWFVQALCS ILE

.:I..: IIIIHIII:IIII.IIIIIII..VDDDMACH . . .KIPVEADFLYAYSTAPGYYSWRNSKDGSWFIQSLCAMLK

EHGKDLE IMQILTRVNDRVARHFESQSDDPHFHEKKQIPCVVSMLTKELY

QYADKLEFMHILTRVNRKVATEFESFSFDATFHAKKQIPCIVSMLTKELY

FSQ 303

FY}i 277

50

35

100

77

150

127

200

177

250

224

300

274

Fig. 3. Colinear alignment of Mch3a and CPP32proteins gaps in the sequence to allow optimalalignment. Amino acid residues are numbered tothe right of each sequence.

Mch3 isoform. The smaller message could be a degradation product oran alternatively spliced Mch3 isoform.

Expression and Autoprocessing of Mch3 in Escherichia coli.We have shown recently that expression of ICE, CPP32, and Mch2ain bacteria as fusion proteins with GST results in processing andformation of active enzymes (6). This process is associated withcleavage of the NFI2-terminal GST prodomain from the active enzyme. The ability to easily purify and estimate the size of the GSTprodomain cleavage product allows prediction of the autoprocessingsite(s). To express Mch3a in E. coli, three GST-Mch3ca expressionvectors were constructed and transformed into DH5cv bacteria. Thefirst construct (GST-Mch3al) encodes GST fused at its COOH ter

minus to amino acids 1—303of Mch3a (Fig. SA). The second construct (GST-Mch3a2) is similar to the first construct; however, it hasan additional 16 amino acids fused in-frame to the COOH terminus ofGST. These 16 amino acids are encoded by bp —48to —1(relative tothe ATG start site) of Mch3a cDNA (see Fig. 1). The third constructencodes GST fused at its COOH terminus to amino acids 24—303ofMch3a (Fig. 5A). After induction with IPTG, bacterial extracts wereprepared from E. coli expressing the recombinant fusion proteins. Theextracts were adsorbed to glutathione-Sepharose resin, washed severaltimes, and then analyzed by SDS-PAGE. As shown in Fig. SB,compared to the GST nonfusion protein control that migrates as a—28-kDaprotein (Fig. SB, Lane GST), the Mch3cxl preparation (Fig.

Fig. 4. Tissue distribution of human Mch3mRNA. Northern blot analysis of human tissuepoly(A@) RNA (2 @xgIlane)using the full-lengthMch3a as a probe. Molecular weight markers inthousands are shown on the left.

7.5—

4.4—

6048

a)

@ Cl)

— a)=-o t°E

a).@3 .@8@ .2@c

@ _o@ L2@cc.2Cl) 0

E ci, Cl)

0@ E

@@@ .@@@@ &ao@ ca

@ a)a)@

@ 0:2 0-@@ 0 Cl)0 o.ca@ 8Ei@

2.4-S

I .3—

Research. on December 26, 2020. © 1995 American Association for Cancercancerres.aacrjournals.org Downloaded from

Table 1 Kinetic Parameters of Mch3a and CPP32

The activity of recombinant Mch3a and CPP32 was measured using bacterial lysatesprepared with ICE buffer [25 mat HEPES-l mat EDTA-5 mM DTT-0.l% CHAPS-lO%sucrose (pH 7-5)1 at room temperature. The Ks for Mch3a and CPP32 were determinedfrom the hydrolysis rate of 50 and 10 psi DEVD-AMC, respectively, after a 30-mmpreincubation of the enzyme with inhibitor. Before incubation with enzymes, purified CnnA was activated by incubation with 5 mat Dli' for 10 mm at37°C.Parameter

Mch3aCPP32Km

(DEVDAMC, ELM) 51 13Km (YVADAMC, ELM) NA >500K (DEVD-CHO, flM) 1.8 0.59K@(YVAD-CHO, gxM) >10 8.5K (Cnn A, @A.M) >1 0.56

CLONING OF Mch3

B

A

pGEX-2T -DLVPRGSPGIHRD

F-'(I)

kDa@@ 1 2 3

4@

30-

20-

14-

pGEX-2T-Mch3al _DLVPRGSI@DDQGCIEEQGVEDSANEDSV@AKPDRSSFVPSLFSKK -‘28. 8 kDa

KKNVTMRSIKTTRDR

pGEX-2T-Mch3a2 -DLVPRGS GLQEFGTRRDCASPSRPTAVGT@DDQGCIEEQGVEDSA -‘31. 1 kDa23

NEDSVDAKPDRSSFVP SLFSKKKKNVTMRSIKTTRDR*

pGZX-2T-Mch3013 _DLVPRGSLAKPDRSSFVPSLFSKKKKNVTMRSIKTTRDR -‘58.4 kDa

Fig. 5. Expression of Mch3a as a GST-fusion protein in E. coli. A, deduced amino acid sequences at the fusion junction between Mch3al, Mch3a2, or Mch3a3 and GST in theirbacterial expression vectors. @,Asp23. The predicted molecular masses of the GST control (pGEX-2T) and the potential GST-prodomain cleavage products are shown to the right ofeach sequence. Metl of Mch3a is circled. Arrow, fusion junction. B, bacterial extracts from cells expressing GST fusion proteins of Mch3al (Lane I), Mch3a2 (Lmie 2), or Mch3a3(Lane 3) were adsorbed to glutatione-Sepharose, washed several times, and then analyzed on a 14% SDS-polyacrylamide gel. The proteins were detected by Coomassie staining. LaneGST, GST purified from cells carrying the pGEX-2T vector. Large arrow, full-length unprocessed GST-Mch3a3 fusion protein; small arrow, processed GST-Mch3a3 that probablylacks its p12 COOH-terminal subunit.

SB, Lane 1) contains a major GST prodomain cleavage product that

migrates as a —30-kDa band. However, because of the additional 16amino acids in GST-Mch3a2, its major GST prodomain cleavageproduct (Fig. SB, Lane 2) migrates as a —32-kDa band. The same—32-kDaGST prodomain product was also obtained when an in vitrotranslated GST-Mch3a2 was incubated with CPP32 or Mch3a (seebelow). Cleavage at Asp23 would generate products that have calculated molecular masses of —28.8and —31.1 kDa (Fig. 5A). Thesesizes are consistent with the —30- and ‘--32-kDaGST-prodomainproducts seen in the GST-Mch3al and GST-Mch3a2 preparations,respectively (Fig. SB, Lanes I and 2). As expected, removal of aminoacids 1—23as in GST-Mch3a3 resulted in expression of a major59-kDa unprocessed GST-Mch3a fusion protein (Fig. SB, Lane 3).Two additional GST-fusion protein products, one migrating as a—47-kDaband and the other as a —29-kDaband, were also produced.The —47-kDa band may represent a partially processed GST-Mch3awithout the COOH-terminal p12 subunit, probably as a result ofcleavage at Aspi 98. The 29-kDa band may represent a cleavageproduct generated by cleavage at a site COOH terminal to Asp23. Thissuggests that Mch3ct can still be cleaved, albeit less efficiently at a siteCOOH terminal to Asp23. Therefore, because our data suggest thatAsp23 and Asp198 are the most likely processing sites, we will referto the large subunit of Mch3cs as p20 and the small subunit as p12.

Kinetic Properties and Enzymatic Activity of Mch3a andCPP32. The kinetic properties of bacterially expressed recombinantMch3a and CPP32 were determined using the peptide substrateDEVD-AMC in a continuous fluorometric assay. The DEVD-AMCsubstrate is the PARP cleavage site Pl—P4tetrapeptide. Both Mch3aand CPP32 exhibited Michaelis-Menten kinetics in cleaving this substrate with Km of S 1 and 13 p.M, respectively (Table 1). The Km ofrecombinant CPP32 (13 p@M)is comparable to the Km of purifiedhuman CPP32 (9.7 ±1.0 pM) reported recently (12). The peptidealdehyde DEVD-CHO is a potent inhibitor of both Mch3a and CPP32at low nM concentrations (KjMch3a 1.8 nM; Kj@p@3@0.59 fl.M).Incontrast, the ICE inhibitor peptide aldehyde YVAD-CHO(KIICE 0.76 nM; Ref. 2) is a very weak inhibitor of both Mch3ca andCPP32 (KIMCh3, > 10 pM; K1@@@32= 8.5 @J.M).The ICE inhibitorcowpox serpin, Crm A (17), is also a very weak inhibitor of Mch3a

and CPP32 (KiMch3a> 1 @.LM‘@iCPP320.56 SM). These data confirmthat the two enzymes, Mch3a and CPP32, are functionally similar toeach other and suggest that they have similar substrate specificity. Thehigh concentration of Cnn A required to inhibit either CPP32 orMch3a (Table 1) suggests that the target of Cnn A inhibition inapoptosis is most probably not CPP32 or Mch3a. In addition, it hasbeen shown recently that Cnn A has 10,000-fold preference for ICEover CPP32 (12). Therefore, we believe that the target(s) of Crm Ainhibition of apoptosis is ICE or an ICE-related protease(s) and notCPP32, as claimed recently (I 1).

CPP32 also has been claimed recently to be the sole PARP-cleavingenzyme in apoptosis (12). However, our data show that Mch3a hasthe same substrate specificity toward PARP as does CPP32. Incubation of purified bovine PARP or human Hela nuclei with Mch3aresulted in a complete cleavage of PARP in less than 15 mm (Fig. 6A).Similar activity was also observed with recombinant CPP32 and withS/M extracts derived from chicken DU249 cells in the committedphase ofapoptosis (Ref. 13; Fig. 6B). Inhibition studies with the serineprotease inhibitors TLCK and TPCK revealed interesting results. At 1mM D'Vf, TPCK was able to inhibit both Mch3a and CPP32 PARP

cleaving activity (Fig. 6C, Lanes 4 and 3, respectively). Under theseconditions, TLCK did not inhibit Mch3a activity but it completelyinhibited CPP32 activity (Fig. 6C, Lanes 3 and 12). In contrast, at 5mM DTT, the inhibition of Mch3a and CPP32 by TLCK and TPCK

was much reduced, although a slight inhibition of CPP32 was ob

6049

94-@“27kDa 67-@

Research. on December 26, 2020. © 1995 American Association for Cancercancerres.aacrjournals.org Downloaded from

CLONING OF Mch3

A

B

Fig. 6. Western blot analysis of PARP cleavageby Mch3a and CPP32. A, cleavage of bovine andhuman PARP by Mch3a. Purified bovine PARP(200 ng; Lanes 2—8)or isolated HeLa nuclei(1 X 106; Lanes 9—IS) were incubated at 37°Cwith buffer (Lanes 2 and 9) or with lysates (50 @xg)from untransformed DH5a E. coli (Lanes 3 and10) or transformed with a cDNA expressingMch3a (Lanes 4—8and 11—15).After the timeindicated above Lanes 2—15,the cleavage productswere analyzed by Western blotting. Mch3a-contaming lysate (50 @zg)incubated for 60 mm in theabsence of PARP was also blotted for comparison(Lane 1). B, cleavage of PARP by CPP32 and S/M

extract. Purified bovine PARP (200 ng; Lanes2—15)was incubated with buffer (Lane 2), withCPP32-containing lysate (30 @xg:Lanes 4—9),andwith S/M extracts (140 g.&g;Lanes 10—15)for thetime indicated. C, effect of DTT concentration onthe sensitivity of Mch3a and CPP32 to TLCK andTPCK. E. coli lysates (30 @xg)containing Mch3a(Lanes 2—7)or CPP32 (Lanes 8—13) were prein

cubated with 0.5% methanol (to control for theeffect of solvent; Lanes 2, 5, 8, and II), with 100gLM TLCK (Lanes 3, 6, 9. and 12), and with 100 psi

TPCK (lanes 4, 7, 10, and 13) for 15 mm at 37°Cin the presence of I (Lanes 2—4and 11—13)or 5(Lanes 5—10)mM DTT. Purified bovine PARP(200 ng) was added to preincubated lysates (Lanes2—13)or to buffer preincubated in the presence of5 m@ D'VF for 15 mm (Lane 1) and was resolvedby immunobloning after 10 mmnof reaction.

PARP

@@ —@ ——‘@@@ .@ PARPfragment

2 3 4 5 6 7 8 9 10 11 12 13 14 15

>1 purIfied bovine PARP

C0@

@@@ CPP32 S/M extract

C.) 60 60 0 5 15 30 60 120 0 5 15 30 60 120(min)

@ =—@ @- - @PARP

__@@ -@ @—@ ——-‘--, .-@ ,@ PARP

fragment2 3 4 5 6 7 8 9 1011 1213 1415

C purifiedbovinePARP

0

00 ()(.) 00 OQ_JQ_ _j 0. @i O@ @JO@I.-,- I- I- I.- I- I—I-

-@ -@ __ -@@ PARP

@@ -@ -@@@ PARPfragment

I 2 3 4 5 6 7 8 9 10111213

Mch3a CPP32

served. This suggests that the concentration of thiol agents mayinfluence significantly the ability of TLCK and TPCK to inhibitcysteine proteases. Furthermore, these drug studies reveal slight butsignificant differences between the closely related Mch3a and CPP32enzymes.

Functional Interchangeability of Mch3a and CPP32 Subunitsin Induction of Apoptosis. To test the ability of Mch3a to induceapoptosis, we took advantage of the ability of baculovirus-expressedICE-like cysteine proteases to induce early apoptosis of infected Sf9cells (5, 6, 8, 9, IS). Sf9 cells were infected with recombinantbaculoviruses encoding full-length Mch3cx or truncated Mch3a orCPP32 variants, separately or in various combinations. Cells werethen examined by light microscopy for morphological signs of apoptosis, such as blebbing of the cytoplasmic membrane, condensation ofnuclear chromatin, and the release of small apoptotic bodies. Inaddition, the genomic DNA was examined for internucleosomal DNAcleavage. Expression of full-length Mch3a in Sf9 cells caused approximately 50% of the cells to undergo apoptosis 42 h postinfection.This was accompanied by induction of internucleosomal DNA cleavage (Fig. 7A, Lane 3). This result was similar to those obtained

previously with ICE, CPP32, and Mch2a (5, 6, 15). In controlexperiments, truncated Mch3a that encodes either the p20 subunit(amino acids 1—198)or the p12 subunit (amino acids 199—303)wereunable to induce apoptosis in Sf9 cells when expressed separately(Fig. 7B, Columns 1 and 2). However, when these two subunits werecoexpressed, —49%of the cells died by apoptosis (Fig. 7B, Column3). Similarly, the two subunits of CPP32 were not apoptotic when

expressed separately (Fig. 7B, Columns 4 and 5) but were apoptoticwhen coexpressed together (Fig. 7B, Column 6). Particularly interesting results were obtained when the Mch3-p20 subunit was coexpressed with the CPP32-pl2 subunit or vice versa (i.e., CPP32-pl7with Mch3-pl2). These combinations were able to cause apoptosis inmore than 50% of the cells (Fig. 7B, Columns 9 and 10). No significant induction of apoptosis was observed in control cells coexpressing Mch3-p20 and CPP32-p17 together or cells coexpressing Mch3-p12 and CPP32-pl2 together (Fig. 7B, Columns 7 and 8). In addition,we have shown recently that coexpression of CPP32 and ICE subunitsin any combination does not induce apoptosis in Sf9 cells (5).

Our data suggest the possibility that Mch3a and CPP32 mayheterodimerize in vivo in mammalian cells to form active proapoptotic

6050

l purified bovine PARP HeLa nuclei

@ @.@ 0

@ Mch3a@ Mch3ocC')@ ________ _5 U) ________@ u1.@

@ 60 60 5 15 30601206060 5 15 30 60120(min)

1mMDTT 5mMDTT 1 mM DTT

Research. on December 26, 2020. © 1995 American Association for Cancercancerres.aacrjournals.org Downloaded from

23

Da DVSr—:@:----T@

Ml

CLONING OF Mch3

A BFig. 7. Induction of apoptosis in Sf@ cells byMch3a and CPP32. A, expression of Mch3a in S1@)cells induces internucleosomal DNA cleavage. Totalcellular DNA was isolated at 42 h postinfection from519 cells infected with the wild-type baculovirus(Lane I) or the recombinant baculoviruses AcNPVMch3a (Lane 2) or AcNPV-ICE (Lane 3). The DNAsamples were analyzed by electrophoresis in a 1.8%agarose gel containing ethidium bromide. Lane M,molecular mass markers. B. Sf9 cells were infectedwith the following recombinantbaculoviruses:Collimo 1, AcNPV-Mch3a-p20; Column 2, AcNPVMch3a-pI2; Column 3. AcNPV-Mch3a-p20 andAcNPV-Mch3a-p12: Column 4. AcNPV-CPP32-p17; Column 5, AcNPV-CPP32-p12; Column 6, AcNPV-CPP32-p17 and AcNPV-CPP32-pI2; Column7, AcNPV-Mch3a-p20and AcNPV-CPP32-pl7;Column 8, AcNPv-Mch3a-p12 and AcNPV-CPP32-p12; Column 9, AcNPV-Mch3a-p20 and AcNPVCPP32-pI2; Column 10, AcNPV-CPP32-p17 andAcNPV-Mch3a-p12. Forty-two h postinfection, cellswere examined microscopically, and several fieldswere counted (average 1500 cells/condition), and thenumber ofapoptotic cells was expressed as a percentage of total cells counted. These data were confirmedusing the DNA cleavage assay as shown in A.

complexes. Inactive antiapoptotic complexes could also be formedbetween the alternatively spliced isoforms of ICE (i.e., ICEs or ICES,Ref. 15) and ICE or ICE-related proteases such as TX and ICEre1@III.Other antiapoptotic complexes could also be formed between Mch2j3,Mch3j3, or ICH- 1s and other closely related members of the family.These possibilities for combinatorial action greatly increase the complexity of the apoptotic response in mammalian cells. To complicatethings further, we have observed thus far that all known mammalian

r@@14$4

60@

40@

2O@

@ -@ -@--f@ @-

0@7 8 9 10

Mch3 cz+ CPP32

123

Mch3 cx

Ced3IICE-like cysteine proteases can exist in a single cell line (in thiscase, human Jurkat T-cell line). The ability of different members ofthe ICE-family such as Mch3 and CPP32 or ICE and TX (18) toheterodimerize suggests that there may be some redundancy in theirfunction. Therefore, it is expected that disruption of the gene of onefamily member such as ICE (19) or CPP32 may not be sufficient toabrogate apoptosis. In addition, because heterocomplexes of cysteineproteases may have specific activities different from those of the

456

CPP32

A XhoI

GST-Mch3cz2

GST-cPP32

CBkDa@ 2 3 4 5 6 123

94-—@@ 67-@@

..‘ — 43-@@=:@I,*.3o@20-

Fig. 8. Cleavage of ProMch3a by CPP32. A, pBluescript vectors containing a GST-Mch3a2 or a GST-CPP32 insert under the T7 promoter were linearized with the appropriaterestriction enzymes as indicated by arrows and then used as templates for in vitro transcription and translation in the presence of l35Sl-methionine. B, Lanes I and 2, the GST-Mch3a2

DNA template was linearized with EcoRI before transcription/translation to allow translation of GST only. and the products of translation were incubated with buffer (Lane 1) or CPP32(Lane 2) for 30 mm at 30°C. The major 28-kDa band in both lanes is GST, and the minor 64-kDa band in Lane 1 is the full-length GST-Mch3a2 fusion protein produced as a resultof incomplete digestion of the DNA template with EcoRI. This 64-kDa band is cleaved by CPP32 in Lane 2 to produce the 32-kDa band seen above the 28-kDa OST band. Lanes 3—6,the GST-Mch3a2 DNA template was linearized with XhoI before transcription/translation, and the product of translation (full-length GST-Mch3a2 fusion protein) was incubated for30 mm on ice with buffer (Lane 3) or at 30°Cwith buffer (Lane 4), CPP32 (Lane 5), or Mch3a (Lane 6). C, the GST-CPP32 DNA template was linearized with EcoRI beforetranscription/translation, and the product of translation (full-length GST-CPP32 fusion protein) was incubated for 30 mm at 30°Cwith buffer (Lane 1), Mch3a (Lane 2). or CPP32 (Lane3). D, the GST-Mch3a2 fusion protein was immobilized on a GST-Sepharose resin, and the resin-GST-Mch3a2 was incubated for I h on ice with buffer (Lane 1) or at 30°Cwith CPP32(Lane 2). The protein products in B and C were analyzed on a 14% SDS-gels, and in D on a 10—20%gradient SDS-gel. Arrows in B and C, cleavage products.

94-67-

43-

20-

14-14-

6051

EcoRI

D12

94-67-43-

30-@

20-14- 1: p11-191: pll-ll

Research. on December 26, 2020. © 1995 American Association for Cancercancerres.aacrjournals.org Downloaded from

CLONING OF Mch3

parental enzymes, this increases the range of their substrates andresults in an extraordinary complexity to the way by which cysteineproteases regulate apoptosis.

Mch3a is a Substrate for CPP32. The ability of Mch3a andCPP32 to autoprocess in bacteria and the high degree of conservationin their structure and substrate specificity raises the possibility thatMch3a might be a substrate for CPP32 or vice versa. To test thispossibility, GST-Mch3a2 and GST-CPP32 fusion proteins were translated in vitro in reticulocyte lysate in the presence of [35S]methionine.The labeled lysates were then incubated with E. coli extracts containing recombinant active CPP32 or Mch3a enzymes (equal DEVDAMC-cleaving activity). After the incubation period, the cleavageproducts were immobilized on GST-Sepharose, washed several times,and analyzed by SDS-PAGE and autoradiography. As shown in Fig.8B, incubation of CPP32 with the in vitro translated GST-Mch3cs2generated the same 32-kDa GST-prodomain cleavage product observed when GST-Mch3a2 was expressed in bacteria (see Fig. SB,Lane 3). This suggests that CPP32 recognizes the DSVD autoprocessing site at position 20—23of proMch3a. Although Mch3a exhibitedsignificantly less activity than CPP32 toward the in vitro translatedGST-Mch3a, a similar product was observed in the Mch3a reaction(Fig. 8B, Lane 6). Intermediate 35-kDa minor GST prodomain cleavage products can also be seen in this reaction. These products arelikely produced by cleavage at a site(s) COOH terminal to Asp23. Nocleavage of GST was observed when CPP32 was incubated with an invitro translated GST control Fig. 8B, Lane 2) or when the in vitrotranslated GST-Mch3a was incubated with buffer (Fig. 8B, Lanes 3and 4). The same experiment was then performed with in vitrotranslated GST-CPP32 (Fig. 8C). In this case, CPP32 showed a veryweak activity toward its precursor and generated a faint GST-prodomain band of 30 kDa as expected from cleavage at Asp28 (Fig. 8C,Lane 3). No cleavage was observed in the buffer control or the Mch3areaction (Fig. 8C, Lanes 1 and 2). Although Mch3a or CPP32 canautoactivate/autoprocess when overexpressed in bacteria, this does notreflect a normal physiological condition, and it may not occur inmammalian cells. Therefore, the ability of CPP32 to cleave theMch3a precursor better than Mch3a itself and the weak activity ofCPP32 or Mch3a toward the CPP32 precursor suggest that Mch3aprecursor may be downstream of CPP32, and that CPP32 is dependenton an upstream protease for activation in vivo. Finally, because CPP32can efficiently cleave the GST-Mch3cs precursor, we decided to purifythe cleavage products and separate them from the GST prodomain.The 35S-labeled GST-Mch3a precursor was immobilized on GSTSepharose and washed several times. The resin-GST-Mch3cs precursor was incubated with active CPP32, and the soluble productscleaved from the immobilized GST-Mch3a precursor were then analyzed on a 10—20%gradient SDS gel and visualized by autoradiography (Fig. 8D). The three bands that migrate as l7—l9-kDa proteinsmay represent the large subunit of Mch3a at different stages ofprocessing. Similarly, the two bands of 12—13kDa size may representthe small subunit of Mch3a. The bands that migrate as 30- and35-kDa proteins may represent Mch3a precursor minus the prodomain.

In conclusion, Mch3 is the first cysteine protease with extensivehomology to CPP32 to be cloned and characterized. The Mch3 geneencodes two Mch3 proteins, an active Mch3a and an inactive Mch3j3splice variant. Because of the high degree of homology between Mch3and CPP32 and their ability to heterodimerize to form active heteromenc complexes, the inactive Mch3@ may function as a dominantinhibitor of both Mch3a and CPP32. The similarity between CPP32and Mch3a in terms of their kinetic properties and their substratespecificity toward the DEVD peptide and PARP provides strongevidence that CPP32 may not be the sole PARP-cleaving enzyme in

apoptosis. The possibility that Mch3a is downstream of CPP32 suggests that CPP32 could be the PARP-cleaving enzyme during theearly stages of apoptosis but that Mch3a may be involved in the finalstages of PARP cleavage and apoptosis. We suggest that activation ofthe death program in mammalian cells is regulated by multiple pathways, and that execution of apoptosis may involve different cascadesof cysteine proteases.

References

1. 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., andBlack, R. A. Molecular cloning of the interleukin-l @3-convethng enzyme. Science(Washington DC), 256: 97—100,1992.

2. Thomberry, 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., Ellison,K. 0., Ayala, J. M., Casano, F. J., Chin, J., Ding, G. i-F., Egger, L. A., Gaffney, E. P.,Limjuco, G., Palyha, 0. C., Raju, S. M., Rolando, A. M., Salley, i. P., Yamin, T-T.,Lee, T. D., Shively, i. E., MacCross, M., Mumford, R. A., Schmidt, i. A., and Tocci,M. i. A novel heterodimeric cysteine protease is required for interleukin-lfi-processing in monocytes. Nature (Lond.), 356: 768—774, 1992.

3. Kumar, S., Kinoshita, M., Nods, M., Copeland, N. G., and Jenkins, N. A. Inductionof apoptosis by the mouse Nedd2 gene, which encodes a protein similar to the productof the Caenorhabditis elegans cell death gene ced-3 and the mammalian IL-l(3-converting enzyme. Genes & Dcv., 8: 1613—1626,1994.

4. Wang, L., Miura, M., Bergeron, L., Zhu, H., and Yuan, i. lch-1, an Ice/ced-3-relatedgene, encodes both positive and negative regulators of programmed cell death. Cell,78: 739—750,1994.

5. Fernandes-Alnemri, T., Litwack, 0., and Alnemri, E. S. CPP32, a novel humanapoptotic protein with homology to Caenorhabditis elegans cell death protein Ced-3and mammalian interleukin-If.3-converting enzyme. i. Biol. Chem., 269: 30761—30764, 1994.

6. Fernandes-Alnemri, T., Litwack, G., and Alnemri, E. S. Mch2, a new member of theapoptotic Ced-3/Jce cysteine protease gene family. Cancer Res., 55: 2737—2742,1995.

7. Faucheu, C., Diu, A., Chan, A. W., Blanchet, A. M., Miossec, C., Herve, F.,Collard-Dutilleul, V., Gu, Y., Aldape, R. A., Lippke, J. A., Rocher, C., Su. M. S-S.,Livingston, D. J., Hercend, T., and Lalanne, J-L. A novel human protease similar tothe interleukin-lfi-converting enzyme induces apoptosis in transfected cells. EMBOJ., 14: 1914—1922, 1995.

8. Kamens, i., Paskind, M., Hugunin, M., Talanian, R. V., Allen, H., Banach, D., Bump,N., Hackett, M., Johnston, C. 0., Li, P., Mankovich, I. A., Terranova, M., andGhayur, T. Identification and characterization of ICH-2, a novel member of theinterleukin-l@-convening enzyme family of cysteine proteases. J. Biol. Chem., 270:15250—15256,1995.

9. Munday. N. A., Vaillancourt, i. P., Au, A., Casano, F. J., Miller, D. K., Molineaux,S. M., Yamin, T. T., Yu, V. L., and Nicholson, D. W. Molecular cloning andpro-apoptotic activity of ICEreIII and ICEreIIII, members of the ICE/CED-3 familyof cysteine proteases. J. Biol. Chem., 270: 15870—15876, 1995.

10. Yuan, J., Shaham, S., Ledoux, S., Ellis, H. M., and Horvitz, H. R. The C. elegans celldeath gene ced-3 encodes a protein similar to mammalian interleukin-1(3-convertingenzyme. Cell, 75: 641—652, 1993.

II. Tewari, M., Quan, L. T., O'Rourke, K., Desnoyers, S., Zeng, Z., Beidler, D. R.,Poirier, G. G., Salvesen, G. S., and Dixit, V. M. Yama/CPP32-@3 a mammalianhomolog of CED-3, is a CrmA-inhibitable protease that cleaves the death substratepoly(ADP-ribose) polymerase. Cell, 81: 801—809, 1995.

12. Nicholson, D. W., Ali, A., Thomberry, N. A., Vaillancourt, i. P., Ding, C. K., Gallant,M., Gareau, Y., Griffin, P. R., Labelle, M., Lazebnik, Y. A., Munday, N. A., Raju,S. M., Smulson, M. E., Yamin, T-T., Yu, V. L., and Miller, D. K. Identification andinhibition of the ICE/CED-3 protease necessary for mammalian apoptosis. Nature(Lond.), 376: 37—43,1995.

13. Lazebnik, Y. A., Kaufmann, S. H., Desnoyers, S., Poirier, G. G., and Eamshaw, W. C.Cleavage of poly(ADP-ribose) polymerase by a proteinase with properties like ICE.Nature (Lond.), 371: 346—347, 1994.

14. Gu, Y., Sarnecki, C., Aldape, R. A., Livingston, D. J., and Su, M. S-S. Cleavage ofpoly(ADP-ribose) polymerase by interleukin-Ib converting enzyme and its homologsTX and Nedd-2. i. Biol. Chem., 270: 18715—18718, 1995.

15. Alnemri, E. S., Fernandes-Alnemri, T., and Litwack, G. Cloning and expression offour isoforms of human interleukin-1f3-converting enzyme with different apoptoticactivities. J. Biol. Chem., 270: 4312—4317, 1995.

16. Kozak, M. An analysis of 5-noncoding sequences from 699 vertebrate messengerRNAs. Nucleic Acids Res., 15: 8125—8132, 1986.

17. Ray, C., Black, R. A., Kronheim, S. R., Greenstreet, 1. A., Sleath, P. R., Salvesen,G. S., and Pickup, D. I. Viral inhibition of inflammation: cowpox virus encodes aninhibitor of the interleukin-If3-converting enzyme. Cell, 69: 597—604, 1992.

18. Gu, Y., Wu, J., Faucheu, C., Lalanne, J. L., Diu, A.. Livingston, D. J., and Su, M. S.Interleukin-I@.3-convening enzyme requires oligomerization for activity of processedforms in vivo. EMBO J., 14: 1923—1931,1995.

19. Li, P., Allen, H., Banerjee, S., Franklin, S., Herzog, L., Johnston, C., McDowell, J.,Paskind, M., Rodman, L., Salfeld, J., Towne, E., Tracey, D., Wardwell, S., Wei, F-Y.,Wong, W., Kamen, R., and Seshadri, T. Mice deficient in IL-lb-converting enzymeare defective in production of mature IL-lb and resistant to endotoxic shock. Cell, 80:401—411,1995.

6052

Research. on December 26, 2020. © 1995 American Association for Cancercancerres.aacrjournals.org Downloaded from

1995;55:6045-6052. Cancer Res   Teresa Fernandes-Alnemri, Atsushi Takahashi, Robert Armstrong, et al.   Related to CPP32Mch3, a Novel Human Apoptotic Cysteine Protease Highly

  Updated version

  http://cancerres.aacrjournals.org/content/55/24/6045

Access the most recent version of this article at:

   

   

   

  E-mail alerts related to this article or journal.Sign up to receive free email-alerts

  Subscriptions

Reprints and

  .pubs@aacr.orgDepartment at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

  Permissions

  Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/55/24/6045To request permission to re-use all or part of this article, use this link

Research. on December 26, 2020. © 1995 American Association for Cancercancerres.aacrjournals.org Downloaded from