Wehavedevelopeda PCRapproachto clonenewapoptoticCed-3/Icelike cystelne protease genes. This approach uses degenerate oligonucleotides encoding the highly conserved pentapeptkles QACRG and GSWFIthat are present In all known apoptodc cysteine proteases. Using thisapproach, we have doned a novel apoptotic gene from human Jurkat Tly@ocytes. The new gene encodes a @34-kIlodaltonprotein that ishighly homologous to human CPP32, Caenorhabditis elegans cell deathprotein CED-3, mammailtan Ich-1 (Nedd2), and mammalian interleukin.lfi converting enzyme. Because of its high homology to the C. elegansCed-3gene,wenamedthenewgenemnmmnlianCed-3homologueMch2.Two Mcb2 transcripts (Mch2a, 1.7 kb; Mch2@3, 1.4 kb) were detected inJurkat T lymphocytesand other cell lines. We believe that the Mch2atranscript encodes the full-length Mch2, whereas the MCh2II transcriptencodes a shorter Mch2 Isoform, probably as a result of alternativesplicing. Like Interleukln-113 converting enzyme and CPP32, recombinantMcb2a, but not MCh2@J,possesses protease activity, as determined by itsability to cleave the fluorogenic peptide DEVD-AMC. CPP32 and Mch2acan alsocleave poly(ADP-ribese) polymerase in vitro, suggesting that theseenzymes participate In poly(ADP-ribose) polymerase cleavage observedduring cellular apoptosis. In addition, overexpression of recombinantMch2a, but not Mch2@, induces apoptoals in Sf9 insect cells. Our datasuggest that Mch2 Is a Ced-3/interleukln-lfl converting enzyme-like cysteine protease and could be another important mediator of apoptosis ininamin@H@n cells.
Introduction
Several members of a new class of cysteine protease genes havebeen discovered recently as regulators of programmed cell death orapoptosis. These genes include mammalian ice3 (1, 2), Ic/i-I (Nedd2)(3, 4), and Cpp32 (5) genes, as well as the Caenorhabditis elegansCed-3cell deathgene (6). Except for ICE, the crystal structureofIch-1, CPP3Z or Ced-3 has not yet been determined.However,basedon structural homology, these enzymes have a similar and uniquestructure that is unrelated to classical cysteine proteases (7, 8). Theyall contain an active site QACRG pentapeptide. Furthermore, structural analysis suggests that these enzymes are synthesized as inactiveproenzymes. The proenzymes are activated by proteolytic cleavage atconserved aspartic acid cleavage sites to generate two polypeptidesubunits. In ICE, these subunits are known as p20 and plO subunitsthatassociate with each otherto form the active heteromericcomplex(2, 7, 8).
Apoptotic cell death is essential for normal development and maintenance of normal tissue size homeostasis in multicellular organisms(9—11).There is growing evidencethat dysregulationof apoptosis
I This work was supported by research Grant Al 35035-01 from the NIH.2 To whom requests for reprints should be addressed, at Department of Pharmacology,
ThomasJeffersonUniversity,BluemleLifeSciencesBuilding,233S. 10thStreet,Philadelphia, PA 19107.
may lead to several human diseases, including cancer and degenerative neuronal diseases such as Alzheimer's and Parkinson's diseases(12—14).Therefore, it is probable that ICE-like cysteine proteases playa significant role in the pathogenesis of these diseases. To isolate andcharacterize novel cysteine proteases, we developed a PCR techniqueto enrich for DNA sequences that encode the highly conserved pentapeptides QACRG and GSWFI present in ICE-like apoptotic cysteineproteases. We were able to clone a second human Ced-3 homologuenamed Mch2.4 The new Mch2 gene is highly homologous to Ced-3and Cpp32 and could be involved in the molecular mechanism ofvertebrate apoptosis.
Materials and Methods
Cloning of Human Mch2. A 10-galaliquot of human Jurkat A Uni-ZAPXR cDNA library (5) containing@ plaque-forming units was denatured at99°Cfor 5 mm and used as a substrate for PCR amplification (30 cycles: 94°Cdenaturation, 55°Cannealing, and 72°Cextension for 45 s at each step). Theprimary amplification was performed with a degenerate primer encoding thepentapeptide GSWFI/GSWYI and T3 vector-specific primer (Stratagene), inthe presence of TaqStart antibody (Clontech) to enhance the specificity andsensitivity of the PCR amplification. A 10-galaliquot of the primary amplification product was then used as a substrate for a secondary PCR amplificationwith two nested primers: a degenerate primer encoding the QACRG pentapeptide and a second vector-specific primer (CAGGAATTCGGCACGAG) located downstream of the T3 primer. This strategy ensures specific amplification of sequences encoding both the GSWFI and the QACRG pentapeptides.The secondary amplification products were blunt ended, phosphorylated, andthen cloned into a SrnaI-cut pBluescript II KS@ vector. All clones werescreened by PCR using ICE- (15), CPP32- (5), and Ich-1- (4) specific primerslocated 5' and proximal to the QACRG sequence. Clones that did not representICE, CPP32, or Ich-1 were then subjected to DNA sequencing using the T3and Ti sequencing primers (Stratagene). This resulted in identification ofseveral Ced-3/ICE-like partial cDNAs. One of these cDNAs with high homology to CPP32 and Ced-3 was then excised from the vector, radiolabeled, andused to screen the original Jurkat A Uni-ZAP XR cDNA library. Fourteen Aclones were purified, rescued into the pBluescript II SK plasmid vector, andthen sequenced.
Construction of Transfer Vectors and Recombinant Baculoviruses.
The Mch2a and f3cDNAs were excised from the pBluescriptII SK vectorwith EcoRl and XhoI restriction enzymes and subcloned into an EcoRI/BgIIIcut pVL1393 after blunting both XhoI and BglII sites. The recombinant transfervectors pVL-Mch2a and pVL-Mch2(3were then used to generate recombinantbaculoviruses as described previously (16, 17).
Expression of Mch2, ICE, and CPP32 in Bacteria and Assay of EnzymeActivity. The Mch2a and @3cDNAs were subcloned in-frame into the BamHl/XhoI sites of the bacterial expression vector pGEX-5X-3 (Pharmacia Biotech,
Inc.). Similarly, ICEy (15) and CPP32 (5) cDNAs were subcloned in-frameinto the BamHI site of the pGEX-5X-3 and pGEX-2T vectors, respectively.Exponentially growing bacteria carrying the expression plasmids were inducedwith 1 mMisopropyl-1-thio-j3-D-galactopyranosidefor 3—6h and then lysed bysonication in a lysis buffer containing 25 m@iHEPES (pH 7.5), 5 mt@iEDTA,2 m@iDTT, and 0.1% 3-[(3-cholamidopropyl)dimethylamino]-l-propanesulfonate. The lysates were centrifuged at 16,000 X g for 10 mm, and the clearbacterial extracts were collected. Fifty-galaliquots of the extracts (2.8 mg/ml
4 The sequences reported in this paper have been deposited in the GenBank data base
(accession numbers U20536 and U20537).
2737
Advances in Brief
Mch2, a New Member of the Apoptotic Ced-3IIce Cysteine Protease Gene Family1
Teresa Fernandes-Alnemri, Gerald Litwack, and Emad S. Alnemri2
Department ofPhar,nacology and the Jefferson Cancer Institute, Thomas Jefferson University. Philadelphia. Pennsylvania 19107
G E E N M T E T®A F Y K R E M F®P A E K Y K M@H R R R G I A L I F N H H R
F F W H LT LP E R RR T C AD RD N L T R RF S D L GF EVK C F N D LKA E
ELLLKIHEVSTVSHADADCFVCVFLSHGEGNHIYAYDAKI
E I Q T L T G L F K G D K C H S L V G K P K I F I I IQ A C R Gi N Q H D V P V IPL®V
V®N Q T E K L®T N I T E V®A A S V Y T L P A G A D F L N C Y S V AEG
Y Y S HRETVN G S WY I Q D L C E ML GK Y G S S L E F T E L L T LVNRK
V S QRR V D F C K D P S A I GK K Q VP C F A S N L T K K LH F F P K S N *
CLONINGOF HUMAN Mch2
total protein) were incubated with 50 gal of the fluorogenic peptide YVADAMC (final concentration, 12 gaM) or DEVD-AMC (final concentration, 50gaM) for different periods of time, and the release of AMC was measured by
spectrofluorometry as described previously (18). For PARP cleavage assay,0.5-gal aliquots of the bacterial extracts were incubated with 120 ng purified
human PARP in a 10-galfinal reaction volume for 20 mm at 37°C.The reactionproducts were then analyzed by SDS-PAGE and Western blotting using thePARP-specific mAb 4C10—5(19).
Results and Discussion
Cloning of Mch2. Using a PCR approach designed to identify andclone novel members of the Ced-3/ICE-like apoptotic cysteine proteases, several partial cDNA sequences were identified; among themwas a new gene with high homology to CPP32 and Ced-3. OthercDNAs with high homology to ICE were also identified (data notshown). The new partial cDNA was used as a probe to screen theoriginal human Jurkat T-lymphocyte cDNA library. This resulted inthe isolation of several cDNA clones. The sequences of two of theseclones are shown in Fig. 1. These two cDNAs are named mammalianCed-3 homologues Mch2ct and Mch2f3. Mch2a contains an openreading frame of 879 bp that encodes a 293-amino acid protein witha predicted molecular mass of —34kDa. The initiator methionine at
nucleotide 79 conforms to the consensus Kozak translation initiationsequence (20). Mch2(3 contains a deletion corresponding to nucleotides 119—385of the Mch2a sequence (amino acids 14—102),and ithas a longer 3' nontranslated sequence (Fig. 1). The deletion inMch2$3 could be due to alternative splicing of the parental Mch2mRNA. Mch2a contains an alternative splice donor/acceptor sitewithin its coding sequence that conforms to the GT/AG rule (Fig. 1;119—385bp). This site is located exactly at the splice junction.Northern blot analysis ofthe expression of Mch2 revealed two mRNAspecies of —1.7and —1.4kb in the human 380 pre-B lymphocytes(Fig. 2, Lane 1) and the human Jurkat T-lymphocytes (Fig. 2, Lane 2).However, there appears to be a difference in the relative level ofexpression of each mRNA species in the two cell lines. We believethat these two mRNA species correspond to Mch2cr and Mch2(3,respectively.
Mch2@3cDNA maintained an open reading frame of 612 bp thatencodes a 204-amino acid protein with a predicted molecular mass of—23kDa. Mch2f3 lacks approximately one-half of its putative p20subunit and is probably inactive. Several alternatively spliced isoforms of ICESwere discovered recently in our laboratory (15). Analternatively spliced Ich-1 isoform (Ich-1 s) was also described re
Fig. 1. Nucleotide and predicted amino acid sequence of the human Mch2a and [email protected] sequence alignment of Mch2a and Mch2(3cDNAs. The predicted amino acidsequence of Mch2a is shown above the nucleotide sequence. The single long open reading frame begins with an ATG at position 79 (M 1) and terminates with TAA stop codon (°)at position 957 (N 293) in Mch2a. Dotted lines, the spliced sequences in Mch2a and @.The putative active site pentapeptide is boxed. The putative aspartate cleavage sites are circled.Amino acid and nucleotide residues are numbered to the right of each sequence.
Expression and Autoprocessing of Mch2, CPP32, and ICE inEscherichia coli. To determine the enzymatic activity of Mch2,CPP32, or ICE, these enzymes were expressed in E. coli as fusionproteins with GST. A GST-CPP32-p20 fusion protein that containsamino acids 1—175of CPP32 and a GST nonfusion protein were usedas controls. After induction with isopropyl-1-thio-f3-D-galactopyranoside, bacterial extracts were prepared from E. coli expressing therecombinant fusion proteins. The extracts were adsorbed to glutathione-Sepharose resin, washed several times, and then analyzed bySDS-PAGE. As shown in Fig. 4A, the ICE'y,CPP32, and Mch2apreparations (Fig. 4A, Lanes 2—4)contain GST-fusion proteins ranging in size from —28—35kDa. The GST nonfusion protein controlmigrates as a -@27—28kDa protein (Fig. 4A, Lane 1). Although thepredicted molecular mass of GST-ICEy fusion protein is —61.5kDa,two bands of -@28and —31kDa can be seen in the ICE'y preparation(Fig. 4A, Lane 2). This suggests that ICEy can autoprocess itself togenerate active ICE by cleaving the NH2-terminal GST-propeptide atone of two Asp cleavage sites. Asp26 of ICE-y (Fig. 4B), whichcorresponds to Asp 119 of ICEct, is a site that is cleaved during ICEactivation (2). Cleavage at this site would generate a GST-fusionprotein with a predicted molecular mass of [email protected] kDa that mightcorrespond to the 31-kDa band (Fig. 4A, Lane 2). Cleavage at Asp 3of ICE'y, although it is not a known ICE cleavage site, could generatethe 28-kDa band (Fig. 4A, Lane 2). Similar to the GST-ICEy preparation, the GST-CPP32 and GST-Mch2a preparations contain smallerthan predicted GST fusion products (Fig. 4A, Lanes 3 and 4). TheGST-CPP32fusion productmigratesas a —29—30kDa protein(Fig.4A, Lane 3). Cleavage at Asp 9 or Asp 28 of CPP32 would generateproducts with predicted molecular masses of —27.3or —29.4kDa,respectively (Fig. 4B). Based on the observed size of the GST-CPP32product (Fig. 4A, Lane 3), we believe that CPP32 is most probablycleaved at Asp 28, although it is possible that both sites are cleavedduring CPP32 autoprocessing. Unlike the GST-CPP32 that containsfull-length CPP32, the GST-CPP32-p20 product that contains a truncated CPP32 migrates as a —46 kDa protein that agrees with itspredicted molecular mass (Fig. 4A, Lane 5). Because this recombinantprotein lacks the p11 subunit (amino acids 176—277),it is inactive anddoes not autoprocess to generate the @-29—30kDa GST-CPP32 cleavage product observed when the full-length CPP32 is used (Fig. 4A,Lane 3). Therefore, if CPP32 is cleaved at Asp 28, based on ourprevious observations (5), this would suggest that CPP32 is made upof two subunits of relative molecular masses of 17 and 11 kDa.However, the exact Asp cleavage sites that are used to generate activeCPP32 remains to be determined by amino acid sequencing. TheGST-Mch2a preparation (Fig. 4A, Lane 4) contains two major bandsthat migrate as —31—32-and —34—35-kDaproteins. This is consistentwith cleavage at Asp 23, Asp 32, or Asp 40 of Mch2a (Figs. 2 and4B). These GST-Mch2a cleavage products are larger than the GSTCPP32 product because of the presence of extra 33 amino acids in theGST-Mch2a fusion constructthatarederivedfromthe 5' untranslatedregion of Mch2a (Fig. 1). A minor band of —27kDa is also presentin this preparation that could be due to cleavage at a site near theCOOH terminus of the GST peptide itself. The majority of GSTMch2g3 was expressed in E. coli in occlusion bodies and was notcleaved (data not shown).
Analysis of the Enzymatic Activities of Mch2, CPP32, and ICEUsing Fluorogenic Tetrapeptides. After establishing that Mch2aand CPP32 can autoprocess in bacteria, we tested their enzymaticactivity using two fluorogenic peptide substrates, YVAD-AMC andDEVD-AMC. The YVAD pentapeptideis the ICE cleavage site inpro-interleukin 1f3 (2), and the DEVD tetrapeptide is a site present inPARP that is cleaved by an ICE-like protein during apoptosis (21).Fig. 5 shows the enzymatic activities of Mch2ca, Mch2@, CPP32, and
2739
—., 1.7 KB
@ 1.4 KB
Fig. 2. Northern blot analysis of human Mch2. Total cellular RNA was isolated from380(Lane1) pm-BlymphocytesandJurkat(Lane2) T-lymphocytes.Equalamountsofthe RNA samples(15 gag/sample)were fractionated on formaldehyde-agarose gel, blottedonto Duralon-UV nylon membrane, and then detected with an Mch2-speciflc probe. Theposition of the 18S rRNA is indicated on the left.
cently (4). These isoforms could regulate the activity of the parentalenzyme by acting as dominant inhibitors. Because active ICE-like cysteine proteases are generated by proteolytic cleavage followed by heterodimerizationof their p20 and plO subunits (2, 7, 8), inactive altematively spliced isofonns could interfere with this process by forminginactive hetemmeric complexes with the parental full-length enzyme.
Mch2 Is a Novel CedIICE-like Cysteine Protease. The predictedMch2u protein sequence is similar to human CPP32 (5), the C.elegans CED-3 protein (6), and mammalian Ich-1 (NEDD2) and ICEproteins (Refs. 1—4;Fig. 3). The full-length Mch2ct protein shows thehighest homology to CPP32. Overall, the two proteins share 38%identity and 56% similarity. However, like CPP32, Mch2a is morerelated to CED-3 than to the remainingcysteine proteases; Mch2ashows 35% identity (56% similarity) with CED-3, 29% identity (52%similarity) with human Ich-1, and 29% identity (52% similarity) withhuman ICE. CED-3, ICE, or Ich-1 are less than 29% identical amongeach other.The predictedstructureof Mch2a appearsto be similartoICE and CPP32 (2, 5, 7, 8). Proteolytic cleavage of Mch2a at Aspl76,Aspl79, Asp186, and/or Aspl93 would generate two polypeptidesequivalent to the p20 and plO subunits of ICE and CPP32 (Fig. 1;Refs. 2, 5, 7, and 8). Mch2a, like CPP32, lacks the long NH2-terminalpropeptide present in other cysteine proteases. However, there arethree potential aspartic acid cleavage sites at positions 23, 32, and 40(Fig. 1) that could be used to remove a short propeptide duringprocessing of Mch2a to the active enzyme. Although Mch2a andCPP32 are equally related to Ced-3, the putative p20 subunit ofMch2cv (amino acids 1—179)is more related to the putative p20subunit of Ced-3 (36% identity) than to the putative p20 subunit ofCPP32 (33% identity; Refs. 5 and 6). Consequently, if the p20 subunitor its equivalent largely determines the enzyme specificity, thenMch2a is more functionally related to Ced-3 than to CPP32.
Fig. 3. Sequence and structural comparison of the human Mch2a with other members of cysteine protease family. Colinear sequence alignment of human Mch2a with human CPP3ZC. elegans CED-3, human Ich-1, and human ICE. Dottedlines. gaps in the sequence to allow optimal alignment. Amino acid residues are numbered to the right of each sequence. Aminoacids identical in at least four of five sequences are boxed. The conserved pentapeptide containing the active site Cys in ICE is boxed and shaded. *, known aspartate cleavage sitesbetween the two subunits of ICE.
ICE'y in total bacterial extracts from cells expressing these enzymes as than CPP32 in cleaving this substrate (Fig. 5A). No detectable enzyGST-fusion proteins, using the YVAD-AMC and DEVD-AMC tet- matic activity was observed with Mch2a or Mch2@ towards thisrapeptides as substrates. Both ICE'y and CPP32 were able to cleave substrate (Fig. 5A). On the other hand, Mch2a (but not Mch2g3),the YVAD substrate, although ICEy was about 3-fold more active ICEy, and CPP32 were able to cleave the DEVD substrate. Interest
Fig. 4. Expression of Mch2, CPP32, and ICE as GST-fusion proteins in E. coli. A. bacterial extracts from cells expressing GST-fusion proteins of ICEy (Lane 2), CPP32 (Lane3), Mch2a (Lane 4), or CPP32-p20 (Lane 5) were adsorbed to glutathione-Sepharose, washed several times, and then analyzed on a 12% SDS-polyacrylamide gel. The proteins weredetected by Coomassie staining. Lane I contains GST purified from cells carrying the pGEX-2T vector. B, deduced amino acid sequences at the fusion junction between Mch2a, CPP32,or ICEy and GST in their bacterial expression vectors. °,potential Asp cleavage sites. The predicted molecular masses of the GST control (pGEX-2T) and the potential GST.clcavageproducts are shown to the right of each sequence. The Asp residues are numbered relative to the first Met (circled) in the nonfusion proteins. Arrow, fusion junction.
Fig. 5. Protease activity of Mch2, CPP32, andICE. Bacterial extracts from cells expressingMch2a, MCh2f3, IcEy, or CPP32 were incubatedwith YVAD-AMC(A)or DEVD-AMC (B) peptidesubstrates for the indicated times. The CPP32 cxtract was diluted 5.fold before incubation with theDEVD-AMC peptide substrate. The release ofAM@was measuredby spectrofluorometryat anexcitation wavelength of 380 ron and an emissionwavelength of 460 am. The concentration of AMCwas determined from a standard curve.
PARP after incubation with recombinant Mch2a, Mch2@3,ICEy, orCPP32, using the 4C10—5antibody. This antibody recognizes anepitope in the 41-kDa COOH-terminal chymotryptic fragment ofPARP(19). CPP32 cleaves PARP to generate a major band of —P90kDa and a minor band of 57 kDa (Fig. 6, Lane 4). The 90-kDa bandis most probably generated by cleaving the DEVD site at residue 214of PARP. We believe that this cleavage product corresponds to the85-kDa PARP cleavage product described by Lazebnik et al. (21) inapoptotic cells. The 57-kDa cleavage product is probably generated bycleavage at a site COOH-terminal to the DEVD site. This product wasnot detected with the C-2-10 antibody used in the previous study (21).This is probably because it recognizes an epitope that is NH2-terminalto the epitope that is recognized by the 4C10-5 antibody used in ourstudy. Mch2a also cleaves PARP to generate a major product of —83kDa and a minor product of —57kDa (Fig. 6, Lane 2) similar to thatobtained with CPP32. PARP is not cleaved by Mch2j3 or ICE'y (Fig.6, Lanes 1 and 3). These data suggest that both CPP32 and Mch2acan cleave PARP. The major cleavage product obtained withMch2a is smaller in size than the one obtained with CPP32,suggesting that the Mch2a cleavage site is COOH-terminal to theCPP32 cleavage site. Furthermore, the deletion in Mch2f3 abrogates its enzymatic activity.
Expression of Mch2a in Sf9 Cells Induces Apoptosis. We haveshown recently that expression of CPP32 or ICE in Sf9 cells resultsin induction of apoptosis within 24—48h after infection (5, 15). Totest whether expression of Mch2ct has a similar apoptotic effect,Sf9 cells were infected with a recombinant baculovirus expressingMch2a or Mch2f3 under the polyhedrin promoter. Cells were alsoinfected with the wild-type virus and the recombinant ICE baculovirus as controls. Morphological, biochemical, and viabilityanalyses revealed that cells infected with ICE or Mch2a, but notwith the wild-type virus or Mch2f3, had several characteristic signsof apoptosis including cytoplasmic membrane blebbing, nuclearfragmentation and condensation, and internucleosomal DNAcleavage (Fig. 7, Lanes 2 and 3). A decrease in viability similar tothat observed previously with cells expressing ICE or CPP32 (5,15) was also observed in cells expressing Mch2a but not Mch2@3(data not shown).
In conclusion, we have cloned a novel apoptotic cysteine proteasenamed Mch2. This was achieved using a PCR approach designed toidentify and clone novel members of the Ced3/ICE-like apoptoticcysteine protease family. The amino acid sequence and predictedstructure of Mch2 is similar to that of ICE and the other members ofthis family such as CED-3, CPP32, and Ich-1. Mch2a and CPP32require an Asp residue in the P1 position of the peptide substrateDEVD-AMC, suggesting that they have a similar substrate require
15 30 60 90 120 150 15 30 60 90 120 150
Time (mm)
ingly, CPP32 is much more active towards this substrate than ICEy orMch2u. Assuming that the bacterial extracts contain a similar amountof each enzyme, we found thatCPP32 is —150-foldmore active thanICE'y or Mch2a in cleaving the DEVD substrate as determined fromthe initial rate of the reactions. The purified GST-fusion productsshown in Fig. 4A or the GST control extract had no enzymatic activitywith either of the substrates(data not shown).
Mch2 and CPP32 Can Cleave PARP. In apoptotic cells, nuclearproteins such as PARP, lamins, and the 70-kDa protein component ofthe Ui small nuclear ribonucleoprotein are cleaved specifically by anunknown ICE-like cysteine protease(s) (21, 22). Cleavage of humanPAR.P at the DEVD site (amino acids 211—214)would generate alarge protein product of predicted molecular mass of 89.3 kDa (aminoacids 215—1014).Fig. 6 shows a Westem blot analysis of human
@-@ Cl
@@ c)@@@ —
kDa
94-
67-
45-
30-
Fig. 6. Western blot analysis of PARP cleavage by Mch2, ICE, and CPP32. HumanPARP was incubated with Mch2@3(Lane 1), Mch2a (Lane 2), ICE-y(Lane 3), or CPP32(Lane 4) at 37°Cfor 20 mist and then analyzed by SDS-PAGE and Western blotting asdescribed under “Materialsand Methods.―Lane 5 contains PARP incubated with a buffercontroL
0.12-Fig. 7. Expression of Mch2a in Sf9 cells induces internucleosomal DNA cleavage.
Total cellular DNA was isolated at 48 h after infection from Sf9 cells infected with thewild-type baculovirus (Lane 1) or the following recombinant baculoviruses: Autographacal(fornica nuclear polyhedrosis virus (AcNPV)-ICE (Lane 2), AcNPV-Mch2a (Lane 3),or AcNPV-Mch2f3(Lane 4). The DNA samples were analyzed by electrophoresis in a1.8% agarose gel containing ethidium bromide. Lane M, molecular mass markers.
ment as ICE. We do not yet know the activators, inhibitors, orsubstrates of ICE-like cysteine proteases. However, our data showclearly that PARP is a substrate for both Mch2a and CPP32. Similarto ICE and Ich-1 (4, 15), the activity of Mch2 might be regulated byalternative splicing. An alternatively spliced isoform, Mch2@3,wasalso isolated. Like Ich-1 s (4), Mch2@3could be a dominant inhibitorof Mch2a and could function as a negative regulator of apoptosis.Alternatively, if this form is cleaved to generate a functional p11subunit, it may then serve as an activator of Mch2. Consequently, thealternatively spliced isoforms of these enzymes may play a criticalrole in their activation or inhibition (15). Tissue-specific regulation ofthe level of expression of these isoforms might be responsible forsensitivity or resistance to induction of apoptosis. The isolation andcharacterization of novel members of this important class of cysteineproteases will enhance the efforts to identify their endogenous substrates and regulators and to design specific drugs that will regulatetheir activity. In addition to the value that will be gained fromunderstanding the molecular mechanism of apoptosis in developmentand homeostasis, characterization of these enzymes will generatesignificant knowledge that could be applied in the treatment of manyhuman diseases such as cancer and many other degenerative diseases.
Acknowledgments
We thankDr. M. Summersfor the baculovirusexpressionsystem and Dr.N. A. Berger for PARP and the 4C10-5 antibody. We also thank Dr. K.Thomaselli for the DEVD-AMC peptide and Dr. L. Wang for technicalassistance.
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