caspases & cancer

7/27/2019 caspases & cancer

1/112004 NaturePublishingGroup

Apoptosis,or programmed cell death, is central to thedevelopment and homeostasis of metazoans15.Dysregulation of apoptosis leads to a variety of humanpathologies including cancer, autoimmune diseases andneurodegenerative disorders610.Since the concept ofapoptosis was established in 1972 (REF. 11), researchefforts have led to the identi fi cati on of hundreds ofgenes that control the ini tiation,execution and regulationof apoptosis in several species3.Compell ing evidenceshows that the mechanism of apoptosis is evolutionarilyconserved.

Caspases are the central components of the apop-tot ic response12,13.Caspases (which are so-named asthey are cysteine proteases that cleave after an aspartateresidue in their substrates14) are a conserved family of

enzymes that irreversibly commit a cell to die.Although the first caspase, interleukin-1-convertingenzyme (ICE;also known as caspase-1),was identifiedin humans15,16, the criti cal involvement of caspases inapoptosis was discovered in the nematode wormCaenorhabdi tis elegans, in which the indispensable geneced-3(cell-death abnormality-3) was found toencode a cysteine protease that closely resembles themammalian ICE17,18.Since then,at least 14 distinct mam-malian caspases have been i dentified, of which thereare 11 in humans12. Caspases and their homologueshave been reported in species that range from thenematode to the dipteranDrosophila melanogaster19

and the lepidopteranSpodoptera frugiperda20,and eventhe yeast Saccharomyces cerevisiae21.

Over the past decade,many key events in caspaseregulation have been documented at the molecular andcellular level3,22.The earlier focus on the genetic and cell-biological characterization of caspases has now beencomplemented by biochemical and structural investiga-tions,giving rise to an unprecedented level of clarity inour understanding of caspase functi on12,2325.I n thisreview,we describe the molecular mechanisms of cas-pase regulation by focusing on the known biochemicaland structural features of caspases and their regulatorsin organisms from fruitflies to humans.

Initiator and effector caspases

Although the fi rst mammali an caspase (caspase-1 orICE) was identi fied as an important regulator of theinflammatory response15,16,at least 7 of the 14 knownmammalian caspases have important roles in apopto-sis12,26.The apoptotic caspases are generally divided intotwo classes: the ini tiator caspases,which includecas-pase-2, -8, -9 and -10 in mammals and Dronc andDredd in fruitf li es; and the effector caspases, whichincludecaspases-3, -6 and -7 in mammals and Drice,Decay, Damm, Dcp1 and Strica in fruit flies (FIG. 1).CED-3 is the only apoptot ic caspase in nematodes andfunctions as both an initiator and an effector caspase.An initiator caspase is characterized by an extended

MOLECULAR MECHANISMS OFCASPASE REGULATION DURINGAPOPTOSIS

Stefan J. Riedl and Yigong Shi

Abstract | Caspases, which are the executioners of apoptosis, comprise two distinct classes, the

initiators and the effectors. A lthough general structural features are shared between the initiator

and the effector caspases, their activation, inhibition and release of inhibition are differentially

regulated. Biochemical and structural studies have led to important advances in understanding

the underlying molecular mechanisms of caspase regulation. This article reviews these latest

advances and describes our present understanding of caspase regulation during apoptosis.

NATURE REVI EWS | MOLECULAR CELL BIOLOGY VOLUME 5 | NOVEMBER 2004 | 8 9 7

Department ofMolecular

Biology,Pr inceton

University, Lewis Thomas

Laboratory,Washington

Road,Pri nceton,New Jersey

08544,USA.

Correspondence to Y.S.

e-mail:

[email protected]

doi:10.1038/nrm1496

R E V I EWS


2/112004 NaturePublishingGroup8 9 8 | NOVEMBER 2004 | VOLUME 5 www.nature.com/reviews/molcellbio

R E V I EWS

PRODOMAIN

The N-terminal amino-acid

sequence of a caspase.Unlike

other proteases,the removal of

the prodomain is not required

for the activation of caspases.

Rather,the presence of the

prodomain is indispensable to

the activation of ini tiator

caspases.

Asp residues that separate the large (~p20) and small(~p10) subuni ts. The p20 and p10 subunits closelyassociate with each other to form a caspase monomer.By contrast, the initiator caspases are auto-activated.Asthe activation of an initiator caspase in cells inevitablytriggers a cascade of downstream caspase activation, it isti ghtly regulated and often requires the assembly of amulti-component complex under apoptotic condi-tions27.For example, the activation of procaspase-9 isfacilitated by theAPOPTOSOME,a ~1.4-MDa complex thatincludesAPAF1(apoptotic-protease-activating factor-1)and cytochromec(see below)27,28.Once activated,theeffector caspases are responsible for the proteolyticcleavage of a broad spectrum of cellular targets,whichultimately leads to cell death.

Intrinsic and extrinsic pathways

In mammalian cells,the apoptotic response is mediatedthrough either the intri nsic pathway or the extr insicpathway,depending on the origin of the death stimuli .The intrinsic pathway is triggered in response to a wide

range of death stimuli that are generated from withi nthe cell,such as oncogene activation and DNA damage.The inactivation of this pathway is generally regarded asa hallmark of cancer7.

The intrinsic pathway is mediated by mitochondria4,and, in response to apoptotic stimuli,several proteins arereleased from the intermembrane space of mitochondriainto the cytoplasm4.Some of the well-characterized pro-teins includecytochromec,SMAC (second mitochon-dria-derived acti vator of caspases)/DIABLO (directinhibitor of apoptosis (IAP)-binding protein with lowpI),AIF(apoptosis-inducing factor),EndoG(endonu-clease G) and OMI/HTRA2(high-temperature-require-ment protein A2;seeFIG.2). Perhaps the most intriguingone of these pro-apoptot ic proteins is cytochromec,which binds to and activates the protein APAF1 in thecytoplasm29. The binding of cytochromecto APAF1induces a conformational change that allows APAF1 tobind to ATP/dATP and to form the apoptosome30,whichmediates the activati on of caspase-9 (REFS 29,3133),thereby triggering a cascade of caspase activation.

The extr insic pathway is initiated by the binding ofan extracellular DEATH LIGAND,such asFasL, to its cell-sur-faceDEATH RECEPTOR,such asFas34.The death l igands areconsti tutively homotr imeric;so binding to their recep-tors leads to the formation of a minimally homotrimericligandreceptor complex that recruits further cytosolic

factors, such as FADD and caspase-8, forming anoligomeric death-inducing signalling complex (DISC)35.Formation of the DISC leads to the activation of the ini-tiator caspase,caspase-8,which then cleaves and acti -vates the effector caspase, caspase-3.The function ofthe DISC in the activation of caspase-8 is thought to beanalogous to that of the apoptosome in the activationof caspase-9, although the detailed molecular mecha-nisms remain unknown. The extri nsic pathway cancrosstalk to the intrinsic pathway through the caspase-8-mediated cleavage ofBID (aBH3-ONLYmember of theBCL2 FAMILY of proteins)36,37,which then triggers the releaseof mitochondrial proteins. For an overview of the

N-terminal region,which comprises one or more adap-tor domainsthat areimportant for its function,whereasan effector caspase usually contains 2030 residues in i tsPRODOMAIN sequence.

All caspases are produced in cells as catalyticallyinactiveZYMOGENSand must undergo proteolytic activa-tion during apoptosis.The activation of an effector cas-pase,such as caspase-3 and Drice, is carried out by aninitiator caspase such as caspase-9 and Dronc,respectively(FIG.2) through cleavage at specific internal

C aspase-3

Effectorcaspases

Initiatorcaspases

C aspase-7

C aspase-6

C aspase-8

C aspase-10

C aspase-9

C aspase-2

Drice

Dcp1

Decay

Damm

Strica

Dredd

Dronc

DED DED

CARD

CARD

DED DED

28 175

23 198

23 179 194

216 374

219 415

385

152 316 331

144 324 351

230

315 331

Effectorcaspases

Initiatorcaspases

~ p20 ~ p10

277

28 217

339

21533 202

323

323

527

255

303

293

479

521

416

CARD

DED DED494

450

435

L1 L2 L3 L4

L1 L2 L3 L4

CED-3

220 374 388

CARD

503

L1 L2 L3 L4

Mammals

Fruitflies

Nematodes

Figure 1 | Apoptotic caspases in mammals, fruitflies and nematodes. The effector and

initiator caspases are shown in red and purple, respectively. C ED -3 (cell-death abnormality-3) is

the only caspase in the nematode worm Caenorhabd itis elegansand so fulfills the role of both

initiator and effector caspase. The position of the first intra-chain activation cleavage (between the

large and small subunits, ~ p20 and ~ p10, respectively) is highlighted by a black arrow, whereas

other sites of cleavage are represented by grey arrows. These other cleavage events are thought

to modulate caspase activity and the regulation of caspases by inhibitor of apoptosis (IA P)

proteins and by other proteins. Unlike other protease zymogens, removal of the N-terminal

prodomain of a caspase is unnecessary for its catalytic activity. The prodomains in initiator

caspases invariably contain homotypic interaction motifs, such as the caspase-recruitment

domain (C AR D) and the death-effector domain (D ED). T he four surface loops (L1L4) that shape

the catalytic groove are indicated. The catalytic residue Cys is shown as a red line at the

beginning of L2. The p20 and p10 subunits together form a caspase monomer. The caspases

and the location of functional segments are drawn to scale.


3/112004 NaturePublishingGroupNATURE REVI EWS | MOLECULAR CELL BIOLOGY VOLUME 5 | NOVEMBER 2004 | 8 9 9

R E V I EWS

ZYMOGEN

The proteolytically inactive

precursor of a protease.

APOPTOSOME

A large protein complex that

comprises cytochromecand

APAF1,and forms in the

presence of ATP or dATP.Theapoptosome recrui ts

pro-caspase-9 and results in the

allosteric activation of caspase-9.

DEATH LIGAND

An extracellular growth factor

that triggers an apoptotic

response in cells.

DEATH RECEPTOR

The cell-surface receptor for the

death ligand.A death receptor

contains an extracellular ligand-

binding domain and an

intracellular death domain.

In mammals, the BH3-only proteins (such as BIDand BIM) transduce the signal to mitochondria afterreceiving apoptotic stimuli. The release of mitochon-drial proteins (such as cytochromec) is mediated byBAK and BAX, the multi domain (that is, containingthe BH1, BH2 and BH3 domains) pro-apoptot icmembers of the BCL2 family of proteins41. APAF1and caspase-9 are the functi onal orthologues of thenematode CED-4 and CED-3 proteins, respectively(FIG.2). In fruit flies, Dark42 (also known as Dapaf-1(REF.43) or Hac-1 (REF. 44)) shares signifi cant sequencesimilarity with the mammalian APAF1 and is impor-tant for the activati on of the init iator caspaseDronc45 (FIG.2). Dronc, in turn, cleaves and activatesthe effector caspase Drice. Dronc and Dr ice shareconsiderable sequence simi lari ty with, and are thefuncti onal homologues of, caspase-9 and caspase-3,respectively (FIG. 2).

Conserved structural features of caspases

Structural information is available for the mammalian

caspase-1 (REFS46,47), -2 (REF.48), -3 (REFS49,50), -7 (REF.51),-8 (REFS52,53) and -9 (REF.54) andSpodoptera frugiperdacaspase-1 (REF.55).Al l i solated caspases were crystal-li zed as homodimers(FIG.3a). The basic structure of acaspase monomer is highly conserved, wi th a large(~20 kDa) and a small (~10 kDa) subunit.Homodimerization is mediated by hydrophobic inter-actions,with six -strands from each monomer form-ing a single contiguous 12-stranded-sheet (FIG. 3a).Several -helices and short -strands are located oneither side of the central -sheet, which gives rise to aglobular fold. The active sites,which are formed by fourprotruding loops (L1L4) from the scaffold, are locatedat two opposite ends of the-sheet (FIG.3a).

Until a few years ago, structural i nformation wasrestricted to an activated caspase that was bound to acovalent inhibitor12 (FIG.3a).Such structures revealedthat the backbone configuration of the active site in allcaspases was highly conserved12 (FIG.3b). Of the fouractive loops,L1 and L4 consti tute two sides of the sub-strate-binding groove, whereas the L3 loop and thesubsequent -hairpin,which are collectively referred toas L3, form the base(FIG.3b). L2 harbours the catalyticcysteine and traverses the groove.Among the four loops,L1 and L3 have a relatively conserved length and com-position among all the caspases,whereas L2 and L4 arehighly divergent.

During caspase activation, the L2 loop is cleavedinto the N-terminal segment,which contains the cat-alyt ic cysteine,and the C-terminal segment,which sta-bil izes the active site of the neighbouri ng monomer.So, the active-site conformati on of one monomer iscri ti call y supported by the L2 loop ( the apostrophedenotes a different monomer) from the neighbouringmonomer12 (FIG.3).

Mechanism of e ffector-procaspase activation

Caspases are synthesized as single-chain zymogens.Aneffector caspase exists consti tutively as a homodimer,both before and after the intra-chain activation cleavage.

intrinsic and extrinsic apoptotic pathways,see the posterby J.C.Reed and Z.Huang entitledApoptosis Pathwaysand Drug Targetsin the online links box.

A conserved cascade of caspase activation

The caspase-activation pathway shows considerablesimilari ty in nematodes, fruitflies and mammals(FIG.2).Genetic studies in nematodes have ident ifi ed fourgenes ced-3, ced-4,ced-9and egl-1 that function

sequenti all y to control the onset of apoptosis1.Theactivation of CED-3 is mediated,at least i n part ,by anadaptor protein, CED-4,which involves the oligomer-ization of CED-4 (REF. 38). In the absence of apoptoticsignall ing,CED-4 is consti tuti vely suppressed by theanti -apoptotic protein CED-9 through direct physicalinteracti ons. Dur ing apoptosis, the negative regula-tion of CED-4 by CED-9 is removed by EGL-1,whichis tr anscripti onall y activated by cell -death sti mul i.CED-9 is a functional and structural homologue ofthe mammalian proteins BCL2 and BCL-X

L39,

whereas EGL-1 is a BH3-only member of the BCL2family of proteins40.

BID, BIM

EGL -1

CED-9

CED-4

CED-3

APAF1

Apaf1

C aspase-9

Dronc

C aspase-3, -7 DriceIAPs

Diap1

BCL2

SM AC /DIABLO C yt c/dATP

ReaperHidG rimSickle

Apoptosis

Apoptosis Apoptosis

C ellular targets C ellular targets

M itochondria

AIF

HTRA2/OM I

EndoG

Apoptotic stimuli

Mammals FruitfliesNemodes

Figure 2 | A conserved apoptotic pathway in nematodes, mammals and fruitflies.

Functional homologues of caspases and caspase regulators across species are indicated by

the same colour. C aspase-9 in mammals and Dronc in the fruitflyDrosophila melanogaster

are initiator caspases, whereas caspase-3 and -7 in mammals and Drice in fruitflies belong

to the class of effector caspases. C ED -3 (cell-death abnormality-3) in the nematode worm

Caenorhab ditis elegansfunctions both as an initiator and effector caspase. The inhibitor of

apoptosis (IA P ) proteins suppress apoptosis by negatively regulating the caspases, whereas

SM AC (second mitochondria-derived activator of caspases)/DIA BLO (direct IAP-binding

protein with low pI) in mammals and the RH G proteins R eaper, H id, G rim and Sickle in

fruitflies can remove the IA P-mediated negative regulation of caspases. AIF, apoptosis-

inducing factor; APA F1, apoptotic-protease-activating factor-1; C yt c, cytochrome c;EndoG , endonuclease G ; HTR A2, high-temperature-requirement protein A2.



R E V I EWS

remain unchanged. However, the active-site loopsundergo drastic conformational changes after cleavage.L2 is twisted, shif ting the catalyti c cysteine from i tsactive conformation (FIG. 4c).Str ikingly, the L2 loop,which provides critical support to the active site of theneighbouring caspase monomer, is fl ipped by nearly180. In addit ion, L3 and L4 become highly f lexible(FIG.4c). These conformational rearrangements in theprocaspase-7 zymogen prevent the formation of a sub-strate-binding groove, thereby explaining why the pro-caspase-7 zymogen does not possess detectable catalyticactivity.

So, the unproductive conformati on of the activesite is a direct consequence of the uncleaved nature ofthe procaspase-7 zymogen,which locks the L2 loop in theclosed conformati on and occludes it from stabilizingthe active site.The intra-chain cleavage allows the L2and L2 loops to switch to their open conformation asobserved in the inhibitor-bound caspase-7 (FIG. 3a).Asthe interactions between L2and L2 and L4 are generallyconserved12, this mechanism is li kely to apply to other

effector caspases.On the basis of this mechanism, theessence of effector-caspase activation is the abil ity ofthe L2 loop to move freely,so as to support the neigh-bouring active site. In this regard, inverting the order ofthe large and small subunits at the primary-sequencelevel effectively frees the L2 loop and is therefore pre-dicted to activate caspases. Indeed, this prediction wasconfirmed for mammalian caspase-3 and -6 (REF.58),aswell as for the fruitfly caspase Drice59.

In addition to the formation of the productiveL2L2 interaction, the intra-chain cleavage allows forthe appropri ate placement of the L3 loop, which isflexible in the zymogen. In the active conformation,the N-terminal region of L3 extends into the cavit ybetween the two caspase monomers.Since this space ispartly occupied by the intra-chain linker in the procas-pase zymogen, it is possible that steric hindrance con-tr ibutes to the inactive conformation of the L3 loop andthe lack of catalytic activity57.

However, the catalyt ic activity of an effector caspase isincreased by several orders of magni tude after suchcleavage.Although we do not yet understand how theinitiator caspases are activated, the mechanism of activa-tion for a representative effector caspase,caspase-7, isrevealed by the conformational changes of the active siteafter the activation cleavage12(FIG.4a).

The active-site conformation before the activationcleavage is revealed by the structure of the unprocessedprocaspase-7 zymogen56,57 (FIG.4b).Compared with theinhibitor-bound active caspase-7 (FIG. 3a), the corestructural elements of the procaspase-7 zymogen

BH3-ONLY

BCL2 homology (BH) domain-3

only.Sequence alignment

among the BCL2-family

proteins has identified four BH

domains,BH1BH4.The BH3-

only members are

pro-apoptotic.

BCL2 FAMILY

A family of proteins that all

contain at least one BCL2

homology (BH) region. The

family is divided into anti-

apoptotic multidomain proteins

(such as BCL2 and BCL-XL),

which contain four BH domains

(BH1,BH2,BH3,BH4), pro-

apoptotic multidomain proteins

(for example,BAX and BAK),

which contain BH1,BH2 and

BH3,and the pro-apoptotic

BH3-only protein family (such

as BID,BIM and PUMA).

L2

L2

L2

L2

L4

L3

L1

L2

L2

L2

L2

L4

L4

L3L4

L1

Inhibitor

L3

L3

L1

L1

(from adjacentmonomer)Cys

Cys

Asp(P4)

Asp(P1)

Val(P2)

Glu

(P3)

a b

Figure 3 | Structural features of caspases. a | T he structure of an inhibitor-bound caspase-7,

which is representative of other caspase structures, is shown. T he caspase core structure is

shown in beige, and the bound peptide inhibitor is shown in a ball-and-stick format. T he four

surface loops (L1L4 and L1L4) that constitute the catalytic groove of each caspase monomer

are labelled and shown in blue. Note that L2 stabilizes the active site of the adjacent caspase

monomer. b | The active-site conformation of all known caspases is conserved. The catalytic

residue C ys, highlighted in red, is covalently bound to the peptide inhibitor. A diagram of the

substrate-binding groove is shown in the upper left corner. L1 and L4 constitute two parallel sides

of the groove, whereas L3 serves as the base. L2, which harbours the catalytic residue Cys, is

positioned at one end of the groove, poised for catalysis. L2 has an important role in stabilizing

the conformation of the L2 and L4 loops. P 1P4 (shown in yellow) represent the four contiguous

residues in the substrate that are recognized by the caspase. This figure was prepared using the

PyM ol molecular graphics system (see the online links box).

L2L2

L2 L2

L2

L2

L4L3

L1

L2

L2L2L2

L2

L4

L4

L4

ba c

L1

L1

L3

L3

L3

Procaspase-7 zymogen

Active caspase-7(bound to inhibitor)

Proteolyticprocessing

Procaspase-7zymogen

Active caspase-7(bound to substrateor inhibitor)

Substrateor inhibitor

Figure 4 | Molecular mechanism of procaspase-7 activation.a | A schematic representation of procaspase-7 activation. T he

active-site loops before and after the proteolytic processing are shown in green and blue, respectively. b | Structure of a procaspase-7

zymogen. T he caspase core structure is shown in beige, and the four surface loops (L1L4 and L1L4) that constitute the catalytic

groove of each caspase monomer are labelled and shown in green. c | The L2 loop has quite a different conformation in the

procaspase-7 zymogen, compared with the inhibitor-bound active caspase-7. T he L2 loop, which is locked in a closed

conformation by covalent linkage, is occluded from adopting its productive and open conformation. U nlike the inhibitor-bound active

caspase-7, the conformation of the active-site loops of the procaspase-7 zymogen does not support substrate binding or catalysis.

This figure was prepared using P yM ol (see the online links box) and M olscript124.



R E V I EWS

Gln residue at the beginning of L2 and an Arg residue atthe end of L3 (REF.26).The Arg residue on L3 also coor-dinates the P3 residue (Glu).The S2 and S4 sub-sitesmap mainly to the L3 and L4 loops; the P2 and P4residues have greater sequence variation12.

Because the backbone configuration of the active sitefor all inhibitor-bound caspases is highly conserved12, itwas once thought that the substrate-binding grooves oncaspases are pre-formed.However, in the structure ofthe free caspase-7, the loops that form the substrate-binding groove are flexible and qui te different fromthose in the inhibitor-bound caspase-7 (REF. 56).Surprisingly, the L2 loop in the isolated active caspase-7sti ll exists in the closed conformation, mimicking theprocaspase-7 zymogen and indicating that the iso-lated active caspase-7 adopts a conformation, which isintermediate between that of the zymogen and that ofthe inhibitor-bound caspase-7 (REF.56).So,binding of aninhibitor or substrate results in the observed conforma-tional switch of the L2 loop from a closed to an openform.This mechanism might not be generally appli ca-

ble to other caspases,as recent structural evidence indi-cates that the active-site conformation of the isolatedactive caspase-3 closely resembles that of the inhibitor-bound caspase-3 (REF.63).Nonetheless, this caspase-3was crystall ized in the presence of excess inhibitors63,and so the scenario of induced conformation cannot beruled out.

Inhibitors of apoptosis

Caspases are subject to transcriptional regulation andpost-translational modifications26. In addition,the con-served IAP family of proteins can potently inhibi t theenzymati c acti vity of active caspases and can perma-nently remove caspases through the ubiquitylati on-mediated proteasome pathway12,64,65.

The IAP proteins,which were originally identified inthe genome of baculovirus on the basis of their abil ityto suppress apoptosis in infected host cell s66,have alsobeen found in mammals and fruit fl ies65,67, but not innematodes.There are eight mammalian IAPs,whichincludeXIAP(X-linked IAP),c-IAP1,c-IAP2,ML-IAP(melanoma IAP)/Livin,ILP2(IAP-like protein-2),NAIP(neuronal apoptosis-inhibitory protein),Bruce/Apollonand survivin; and two fruitf ly IAPs, which includeDiap1 (also known asThread) and Diap2 (FIG.5). Inmammals,caspases-3, -7 and -9 are subject to inhibitionby IAPs12 (FIGS 2,5). Interestingly, although caspase-9

binds to several IAPs, it is primari ly inhibi ted by XIAP.By contrast,caspase-3 and -7 are inhibited by XIAP and,to a lesser extent,by c-IAP1,c-IAP2 and NAIP(REFS65,68).In frui tflies,Diap1 directly inhibits the catalytic activityof Drice and targets Dronc to the ubiquitylation-medi-ated degradation pathway61,69.

The hallmark of IAPs is the baculoviral IAPrepeat (BIR) domain,an ~80-amino-acid zinc-bindingdomain70,71.XIAP,c-IAP1 and c-IAP2 contain three BIRdomains each,and the different BIR domains have dis-tinct functions12(FIG.5). In XIAP, the third BIR domain(BIR3) potently inhibits the activity of processed cas-pase-9,whereas the linker region between BIR1 and

Mechanisms of substrate recognition

Caspases recognize at least four contiguous aminoacids in their substrates,P4-P3-P2-P1,and cleave afterthe C-terminal residue (P1), which i s usuall y an Aspresidue12 (FIG.3b).Although caspases were thought tohave an exclusive specificity for Asp, Dronc auto-processes after a Glu residue60and efficiently cleaves thefruitfly IAP Diap1 (see below) after a Glu residue61.Thepreferred P3 position is Glu for all caspases that havebeen examined12,62. The preference in the P4 positi onvaries among different groups of caspases and con-tr ibutes to their substrate specificity.

The sequence specificity of caspase substrates isdetermined by the four active-site loops.The bindingpockets for the P4-P3-P2-P1 positions in the substrateare known as the S4-S3-S2-S1 sub-sites, respectively.Str uctural studies using covalent peptide inhibitorshave shown that these pockets are located mostlybetween the base (L3) and the two sides (L1 and L4) ofthe substrate-binding groove12.The S1 and S3 sub-sitesare nearly ident ical among all caspases, whereas the

location of the S2 and S4 sub-sites is conserved12.TheP1 residue (Asp) is coordinated by three invariantresidues at the S1 sub-site,an Arg residue from L1, a

Fruitflies

Bruce/Apollon

ILP2

M L-IAP /Livin

Survivin

NAIP

c-IAP1

c-IAP2

XIAP

Diap1

Diap2

BIR

BIR R ING

BIR

BIR

BIR1 BIR2 BIR3

RING

BIR1 BIR2 BIR 3 R ING

BIR1 BIR2 BIR3 C AR D R ING

BIR1 BIR2 BIR3 C AR D RING

BIR1 BIR2 BIR3 RING

BIR 1 BIR 2 RING

C aspase-3, -7 C aspase-9

Drice Dronc

Mammals4,845

1,403

142

298

236

604

618

497

438

498

Figure 5 | IAPs in mammals and fruitflies.Proteins of the inhibitor of apoptosis (IA P) family

include XIAP (X-linked IAP), c-IAP 1, c-IAP2, ILP2 (IAP -like protein-2), M L-IAP (melanoma

IAP)/Livin, N AIP (neuronal apoptosis-inhibitory protein) and survivin, and are also known as

M IHA/ILP1, M IHB/HIAP 2, M IHC /HIAP 1, Ts-IAP, K IAP, B IRC 1 and TIA P, respectively. A conserved

linker peptide that precedes the BIR2 (baculoviral IAP repeat-2) domain of XIAP, c-IAP1 or c-IAP 2

(shown in red) is responsible for inhibiting caspases-3 and -7 in mammals. O n the basis of

structural information, residues 126143 (FLLN K DV G NIAK YDIRVK ) of NA IP are predicted to carry

out this function, as indicated in red. O nly the BIR 3 domain of XIAP can potently inhibit caspase-

9. In fruitflies, the BIR1 and BIR2 domains of Diap1, but not Diap2, are responsible for inhibiting

the caspase-3 and -9 homologues Drice and D ronc, respectively. T he biochemically

characterized BIR domains that have known functions are highlighted in colour, whereas other

domains in the various IAP s are shown in grey. CARD, caspase-recruitment domain.



R E V I EWS

Although the li nker peptide that precedes the BIR2domain of XIAP has an important role in inhibiting cas-pase-3 or -7,this fragment is insufficient in isolation70,77.However,an engineered protein with the linker peptidefused either N- or C-terminal to BIR1 was ful ly able tobind to and inhibit caspase-3,whereas neither BIR1 norBIR2 showed any inhibit ion in i solation70.These obser-vations indicate a scenari o in which the linker peptideneeds to be presented in a competent conformation by asurrounding domain. In support of this hypothesis,thelinker peptide fused to glutathione-S-transferase (GST)inhibited caspase-3 or -7 (REFS77,78).Nonetheless, theBIR domains also contr ibute to the inhibi tion of cas-pases,as the full-length XIAP shows a higher potencythan the fusion protein between GST and the linkerpeptide77,78. Consistent with this observation, theBIR2 domain of XIAP also makes direct interactionswith caspase-3(REF.76).

IAP-mediated inhibit ion of caspase-9. Onlyprocessed caspase-9 is subject to inhi bi tion by the

BIR3 domain of XIAP. Mutational analysis identi fiedthree residues of XIAP Trp310, Glu314 andHis343 that are indispensable to XIAP-mediatedinhibi tion of caspase-9 (REF. 79). However, these threeresidues define two distinct surface areas in the BIR3structure.Trp310 and Glu314 li ne the inner side of aconserved surface groove, whi ch has been shown toform the binding site of the N-terminal tetrapeptideof the small subunit of caspase-9. This interaction i sessential to XIAP-mediated inhibit ion of caspase-9.On the other hand,Hi s343 is located on the oppositeside of BIR3.Mutation of His343 to Ala in XIAP didnot affect binding to caspase-9 but did abolish inhi-bition79. So, binding by XIAP is necessary but notsuff icient for the inhibi ti on of caspase-9. Thesemutati onal analyses further indi cate two separatebinding interfaces between caspase-9 and the BIR3domain of XIAP.

A mechanistic explanati on for the inhibit ion ofcaspase-9 by XIAP was revealed by the crystal structureof caspase-9 bound to the BIR3 domain of XIAP(REF. 80) (FIG. 7a). In the uninhibited state,the processedcaspase-9 is present exclusively as a monomer80.Thiscaspase-9 monomer has the potential to be acti vatedby the apoptosome,as well as inhibited by XIAP.Theinteraction between the conserved surface groove ofthe BIR3 domain of XIAP and the N-terminal

tetrapeptide of the small subunit of caspase-9 anchorstheir mutual recognit ion80,81; the BIR3 domain usesanother surface patch, the one defi ned by His343, toheterodimerize with a caspase-9 monomer throughthe same interface that is required for the homodimer-ization of caspase-9 (REF.80) (FIG.7a).This trapped cas-pase-9 monomer adopts a catalyticall y incompetentconformation at the active site80 (FIG.7b). So, XIAPinhibits caspase-9 by sequestering it in a monomericstate,which prevents catalytic activity.

The specific recognit ion of the caspase-9 dimeriza-tion interface requires four amino acids (includingHis343) in the BIR3 domain of XIAP, which are not

BIR2 specifi cally targets caspase-3 and -7 (REF. 12).Survivin72,which contains a single BIR domain,does notinhibit caspase activityin vitro.Another single-BIR-con-taining IAP,ML-IAP/Livin,was reported to inhibit bothcaspase-3 and -9(REFS7375),although it does not seem tocontain the sequence elements that are required for thisinhibition. In frui tfl y Diap1,the second BIR domain(BIR2) is essential for the negative regulation of Dronc,whereas BIR1 is responsible for inhibiting Drice61,69.

Mechanisms of caspase inhibition

IAP-mediated inhi bit ion of effector caspases.The mole-cular mechanism of IAP-mediated inhibition of effectorcaspases is revealed by the crystal structures of caspase-3(REF. 76) and caspase-7 (REFS 77,78) bound to aninhibitory XIAP fragment. In these structures,a shortlinker peptide that precedes the BIR2 domain ofXIAP forms identical interactions with caspase-3 or -7(FIG. 6a). Compared with the covalent peptideinhibitors, the linker peptide of XIAP occupies theacti ve site of caspase-3 or -7 in a reverse orientation,

which results in a blockade of substrate entry (FIG.6b).Asp148 of XIAP,which was shown to be essential forthe inhibit ion of caspase-3 (REF.70),binds the S4 pocketin a manner simil ar to the P4 residue (Asp) of thecovalent peptide inhibitors.In addit ion, Val146 makesa similar set of van der Waals contacts to surroundingcaspase residues,as does the P2 residue. In contrast tothe covalent pepti de inhibi tors, Gly144 of XIAP islocated close to the S1 pocket within van der Waalscontact distance of the catalytic cysteine in the caspases.The structural information not only allows the identi fi-cation of crit ical inhibiti ng residues in c-IAP1 andc-IAP2,but also predicts that residues 126143 of NAIPare responsible for inhibiting caspase-3 and -7(FIG.5).

L2

L2L4

L3

L1

L2

L2

L4C

C

N

a b

L1

L3

Caspase-7

Caspase-7XIAP

XIAPAsp148

Val147

Gly144

Val146

BIR2

Figure 6 | Molecular mechanism of IAP-mediated inhibition of effector caspases.a | The

crystal structure of caspase-7 (shown in beige and cyan) bound to an XIAP (X-linked inhibitor of

apoptosis (IAP)) fragment (orange). The interactions primarily occur between a linker segment that

is N-terminal to the BIR 2 (baculoviral IAP repeat-2) domain of XIAP and the active site of caspase-7.

b | A close-up view of the active site of caspase-7 (in a surface representation) bound to an XIA P

fragment (orange). Two hydrophobic residues of XIA P, Val146 and Val147, make several van der

Waals interactions with a conserved hydrophobic pocket on caspase-7. Asp148 of XIA P, which

occupies the S4 substrate-binding pocket, forms hydrogen bonds with neighbouring residues in

caspase-7. These interactions are also conserved in the caspase-3XIAP complex76. T his figure

was prepared using PyM ol (see the online links box) and G RASP 125.



R E V I EWS

this bond is protected by the neighbouring N terminus ofp35,which occludes access by water molecules85(FIG.8b).Another baculoviral protein, p49,functions simi larly top35 (REFS8688). In addition, theSERPIN CrmA,which isderived from the cowpox virus,has also been shown toinhibit several caspases,probably through the disrup-ti on of the active site of the enzyme as was shown forthe inhibit ion of serine proteases by serpins89.

A tetrapeptide IAP-binding motif

During apoptosis,the IAP-mediated inhibition of cas-pases is antagonized by a family of proteins that containan IAP-binding tetrapeptide moti f90 (FIG. 9a). In mam-mals,the founding member of this family is the mito-chondrial protein SMAC/DIABLO91,92. Due to thecleavage of the mitochondria-targeting sequence, themature SMAC/DIABLO protein contains a tetrapep-tide at i ts N terminus Ala-Val-Pro-I le which bindsto the conserved surface groove on the BIR3 domain ofXIAP(REFS93,94).During apoptosis,SMAC/DIABLO isreleased from the intermembrane space of mitochondria

into the cytosol,where it interacts with several IAPs andremoves the IAP-mediated inhibi tion of both ini tiatorand effector caspases95.Another mi tochondrial protein,HTRA2/OMI,also contains an IAP-binding tetrapeptidemotif at its N terminus (FIG. 9a) and can antagonizeXIAP-mediated inhibition of caspase-9 at high concen-trations96100. However, the bovine homologue ofHTRA2/OMI lacks this moti f101,which calls into ques-tion whether HTRA2/OMI can antagonize XIAP inhibi-tion under physiological settings.

In fruit fl ies, there are at least four functionalhomologues of SMAC/DIABLO: Reaper, Hid, Grimand Sickle, which are collectively referred to as theRHG PROTEINS. The RHG proteins bind to and antago-nize Diap1-mediated suppression of Dronc andDrice. Similar to SMAC/DIABLO,the RHG proteinscontain an N-terminal IAP-binding tetrapeptidemotif(FIG. 9a).Another frui tf ly protein, Jafrac2, con-tains a divergent tetrapepti de motif(FIG. 9a), but hasnonetheless been shown to bind to Diap1 and tocounter Diap1-mediated Dronc suppression102. Aswill be discussed below, the IAP-binding tetrapeptidemoti f that is found in caspase-9, SMAC/DIABLO andthe RHG proteins has an essential function in the reg-ulation of caspase function.

The binding site for thi s conserved tetrapepti demotif is the conserved surface groove on the BIR2 and

BIR3 domains of XIAP (REFS93,94), c-IAP1 and c-IAP2,or on the BIR1 and BIR2 domains of Diap1(REFS61,103).Such recognition,which has been documented in mole-cular detail (FIG.9b), requires an invari ant N-terminalAla residue,with its methyl side chain fitt ing into a con-served hydrophobic pocket and i ts main-chain groupsmaking hydrogen bonds to conserved surface residuesin IAPs.Mutation of Alato any other amino acid,such asMet or Gly,results in the abrogation of interactions withthe BIR domains94,95. The next three residues of thetetrapeptide moti f can tolerate some vari ation butstrongly prefer Pro in the thi rd positi on and a bulky,hydrophobic residue in the fourth position (FIG.9a).

conserved in c-IAP1 or c-IAP2(REF.80).This observationexplains why c-IAP1 and c-IAP2 can bind to,but do notinhibit,caspase-9.

Covalent inhibit ion of caspases by viral proteins. In con-trast to XIAP,which specifically targets the mammaliancaspases-3, -7 and -9, the baculoviral p35protein potentlyinhibits most caspases,both in vivoand in vitro66.Caspaseinhi bit ion by p35 correlates with the cleavage of it sreactive-site loop after residue Asp87,fol lowed by thesubsequent formation of a covalently li nkedproteaseinhibitor complex8284. In parallel, the N ter-minus of p35 is translocated into the active site of thecaspase85. The crystal structure of caspase-8 in com-plex wi th p35 shows that the catalytic residue Cys360of caspase-8 is covalentl y linked to Asp87 of p35through a thioester bond85 (FIG. 8a). Although athioester bond i s generally susceptible to hydrolysis,

SERPIN

A family of serine-protease

inhibitors.Serpin inactives

protease by deformation of the

active site.

RHG PROTEINS

Named after the frui tfly Reaper,

Hid and Grim proteins.The

name now refers to a larger

family ofpro-apoptotic proteins

in fruitflies that share an

N-terminal inhibitor of

apoptosis (IAP)-binding

tetrapeptide motif.

L2

L2

L4

ba

L1

L1

L3

L3

L3L4

L2

C aspase-9 (bound to BIR 3 of XIAP )

C aspase-9 (inactive)

C aspase-9 (active)

Caspase-9

BIR3 of XIAP

Figure 7 | Molecular mechanism of XIAP-mediated inhibition of caspase-9.a | S tructure of

active caspase-9 bound to the BIR 3 (baculoviral IAP repeat-3) domain of XIA P (X-linked inhibitor

of apoptosis (IAP)). The BIR3 domain binds to a large caspase-9 surface that is normally required

for its homodimerization. C aspase-9 and the BIR 3 domain of XIAP are shown in beige and green,

respectively. T he active-site loops are shown in dark blue. The catalytic residue, C ys287 on loop

L2, and the zinc atom in the BIR 3 domain of XIA P are shown in red. b | S uperposition of the four

active-site loops of the BIR3-bound caspase-9 (blue) and the active (yellow) and inactive (purple)

monomers of the caspase-9 homodimer. The active-site conformation of the B IR3-bound

caspase-9 closely resembles that of the inactive caspase-9 monomer. This figure was prepared

using PyM ol (see the online links box) and M olscript124.

ba

Asp87

Cys360

p35 p35p35

C aspase-8

Caspase-8

N-terminal loop

Reactive-site loop

N

Figure 8 | Molecular mechanism of p35-mediated inhibition of caspase-8.a | S tructure of

caspase-8 (shown in blue and green) bound to the baculoviral caspase-inhibitor protein p35

(shown in orange and purple). O ne molecule of p35 binds to each monomer of the caspase-8

dimer. b | C lose-up view of the covalent inhibition of caspase-8 by p35. The thioester intermediate

is shown between Asp87 of p35 and Cys360, which is the active-site residue, of caspase-8. The

N terminus of p35 restricts solvent access to this intermediate. This figure was prepared using

M olscript124.



R E V I EWS

Dronc59,102,107110.The N-terminal IAP-binding motifs ofthe RHG proteins bind to a conserved surface grooveon the BIR2 domain of Diap1(REF.103),which indicatesthat Dronc might bind to Diap1 in a way that is similarto the caspase-9XIAP interactions.However, Droncdoes not contain any sequences that resemble the IAP-binding tetrapeptide moti f69. Int riguingly,biochemicalanalyses revealed that the BIR2 domain of Diap1 recog-nizes a 5-amino-acid sequence in the linker regionbetween the prodomain and the caspase unit ofDronc69. This recogniti on is essential for the Diap1-mediated negative regulation of Dronc69.Remarkably,structural analysis revealed that the Dronc-binding sur-face on the BIR2 domain of Diap1 coincides with thatrequired for binding to the N-terminal sequences of theRHG proteins69 (FIG.10). This explains how the RHGproteins competitively eliminate Diap1-mediated nega-tive regulation of Dronc.

The molecular mechanisms between IAP-mediatedregulation of caspase-9 and Dronc are different not onlyin their mutual recogniti on but,more importantly, in

the way that the caspases are regulated. In mammals,XIAP potently inhibits the catalytic activity of caspase-9(REF.70). In fruitfli es,however,Diap1 shows no effect onthe catalytic activity of Dronc61.Rather,Diap1 functionsas an E3 ubiqui tin l igase that recognizes and ubiquity-lates Dronc69,105, thereby targeti ng Dronc for protea-some-mediated degradation.

Removal of effect or-caspase inhibition

Caspase-3 and -7 regulati on by IAPs and SMAC/DIABLO.

Although the SMAC/DIABLO tetrapeptide in isolationcan remove XIAP-mediated caspase-9 inhibition95,it isnot capable of removing the IAP-mediated inhibition ofeffector caspases.The reasons are simple: the binding sitefor the tetrapeptide motif maps to the surface groove ofthe BIR2 or BIR3 domain of XIAP (or c-IAP1 or c-IAP2),whereas the fragment that is responsible for inhibitingcaspase-3 or -7 is located between the BIR1 and BIR2domain of XIAP (or c-IAP1 or c-IAP2).Although a con-clusive mechanism remains elusive,modelling studiesindicate that steric clashes preclude XIAP from simulta-neously binding to caspase-3 and SMAC/DIABLO77,111.Importantly,binding to both the BIR2 and BIR3 domainsis required for the SMAC/DIABLO-mediated removal ofcaspase-7 inhibition111.This steric clash precludes XIAPfrom simultaneously binding to caspase-7 andSMAC/DIABLO. In this model, binding to the BIR

domains requires not only the N-terminal tetrapeptide ofSMAC/DIABLO,but also an extensive surface that isavailable only in the wild-type dimeric SMAC/DIABLOprotein77.This model is consistent wi th the observationthat monomeric SMAC/DIABLO mutants only weaklyinteracted with the BIR2 domain and were unable toremove the IAP-mediated caspase-3 inhibit ion95.

Dr ice regulat ion by Di ap1 and RHG proteins.Drice isthe caspase-3 orthologue in fruitfli es. In mammals,theinhibiti on of caspase-3 or -7 entails a linker segmentthat immediately precedes the BIR2 domain of XIAP,c-IAP1 or c-IAP2.However, this li nker sequence is not

Removal of initiator-caspase inhibition

Caspase-9 regulat ion by XIAP and SMAC/D IABLO.

During apoptosis,SMAC/DIABLO in mammals and theRHG proteins in fruitflies antagonize IAP-mediated inhi-bition of both the initiator and effector caspases throughdistinct mechanisms,which both require physical interac-tions with IAPs(REF.12).The N terminus (Ala-Thr-Pro-Phe) of the small subunit of caspase-9 conforms to theIAP-binding tetrapeptide motif81 (FIG. 9). Subsequentexperiments confirmed that this sequence is indeed pri-mari ly responsible for the interactions between caspase-9and XIAP (REF.81) (FIG.9b).Before proteolyt ic processing,the procaspase-9 zymogen does not stably interact withIAPs.Proteolytic cleavage of procaspase-9 after Asp315results in the exposure of an internal tetrapeptide moti f,which recrui ts XIAP to inhibit caspase-9.During apopto-sis,SMAC/DIABLO is released from mitochondria intothe cytoplasm, where it uses a similar IAP-bindingtetrapeptide motif to bind to the BIR3 domain of XIAP(FIG.9b),thereby competitively removing caspase-9(REF.81).So, a conserved IAP-binding moti f in caspase-9 andSMAC/DIABLO mediates opposing effects on caspase

activity.

Dronc regulation by D iap1 and RHG proteins.Given theextraordinary conservati on of the apoptotic pathwaybetween mammals and frui tflies(FIG.2), it was anticipatedthat the underlyi ng mechanisms of caspase regulationshould be conserved.Surprisingly,however, recent bio-chemical and structural studies have revealed differentmechanisms for the regulation of caspases in fruitflies61,69.

Dronc is the caspase-9 orthologue in fruitflies.Simi lar to the situation in mammals, Diap1 directlybinds to and suppresses Dronc104106,whereas the RHGproteins can relieve Diap1-mediated suppression of

SM AC/DIABLO (AVPI)bound to BIR3 of XIA P

C aspase-9 (ATP F) bound toBIR 3 of XIAP

Ile

Pro

Val

Ala Ala

Phe

Pro

Thr

baAVPIAQKS

ATPFQEGL

AVPYQEGP

ATPVFSGE

AVPSPPPA

AVPAPPPT

AVAFYIPD

AIAYFLPD

AVPFYLPE

AIPFFEEE

AKPEDNES

SM AC/DIABLOHuman caspase-9M ouse caspase-9

Xenopus laeviscaspase-9Human HTR A2/O M IM ouse HTR A2/O M I

Reaper

Vertebrates

Fruitflies

G rim

HidSickleJafrac2

Figure 9 | SMAC/DIABLO-mediated removal of caspase-9 inhibition by XIAP.a | The

inhibitor of apoptosis (IA P )-mediated inhibition of caspases can be antagonized by a group of

proteins including SM AC (second mitochondria-derived activator of caspases)/D IABLO

(direct IAP -binding protein with low pI), caspase-9 and HTRA2 (high-temperature-requirement

protein A2)/O M I in mammals and the RH G proteins Reaper, H id, G rim and Sickle in the fruitfly

Drosophila melanogaster that contain a conserved IAP -binding tetrapeptide motif. The

tetrapeptide motif has the consensus sequence A-(V/T/I)-(P /A)-(F/Y/I/V/S ), and the fruitfly

proteins contain another binding component (conserved residues 58). P referred and allowed

amino acids are indicated in purple and yellow, respectively. Further conserved amino acids in

fruitflies are shown in blue. b | M olecular mechanism of SM AC /DIA BLO -mediated removal of

caspase-9 inhibition by XIA P (X-linked IAP). The IAP -binding tetrapeptide motifs from

SM AC /DIA BLO (AVP I) and from caspase-9 (ATPF) both bind to the same conserved surface

groove on the B IR 3 (baculoviral IAP repeat-3) domain of XIAP. It is this mutual exclusion that

allows SM AC/DIABLO to remove the XIAP-mediated inhibition of caspase-9. This figure was

prepared using P yM ol (see the online links box).



R E V I EWS

also in the removal of caspase inhibit ion. So,this con-served IAP-binding tetrapeptide moti f helps to uni fymechanisms of apoptosis regulation from fruitf li es tomammals.

Acti vation of ini ti ator caspases.Our understanding ofhow the ini tiator caspases are activated is far from com-plete113.Unli ke the effector caspases, for which both thezymogen and the activated enzymes exist as a constitu-tive homodimer, the case of the ini tiator caspases seemsto be a compli cated one.Caspase-9, both before andafter proteolytic processing,is present predominantly asa monomer54,80. Caspase-8, on the other hand, wasreported to be present in an equilibrium betweenmonomers and dimers114,115.These physical differencesdetermine their functional variations.The fact that cas-pase-9 is a monomer predicts that i ts active site wouldbe in an incompetent conformation,due to the absenceof the supporting L2 loop. Restor ing the competentconformati on of the active site is probably importantfor caspase-9 activati on. In cell s, this is accomplished

through association with the apoptosome,which resultsin a dramatic increase (up to 2,000-fold) in the catalyticactivity of caspase-9 (REF.31,81).

Unfortunately,we do not yet understand how cas-pase-9 is activated by the apoptosome.Two models havebeen proposed.The induced-proximi ty model statesthat the initiator caspases auto-process themselves whenbrought into close proximi ty of each other116. Thismodel is a succinct summary of experimental observa-tions in which accelerated processing and elevated cas-pase acti vi ty were detected after fusion of the targetcaspase with a heterologous dimerization domain38,117120.However, this model merely descri bes a process, anddoes not address the mechanism of ini ti ator-caspaseactivation. The proximity-induced dimerizationmodel121 which represents a quali tative refinement ofthe induced-proximity model argues that the cas-pase-9 and caspase-8 ini tiator caspases are activated ondimerization,which is facili tated by the apoptosome andDISC ol igomeric complexes, respectively54,114,115.Theessence of this model is that,on dimerization,the missingL2 loop is provided; this, together with the supportinginteractions from the dimer interface, restores the com-petent conformation of the active site.However,as thepurpose of the L2 loop i s to regulate allostericall y theactive-site conformation, this might be accomplished bythe apoptosome by entirely different means28,113.At pre-

sent, the accuracy of this model awaits further testing,asthe supporting evidence is not definitive.

Caspase regulat ion in fr ui tf li es and nematodes.Ourknowledge of programmed cell death pr imarily comesfrom investigations of several model organisms thenematode, fruitfl ies and mammals.The apoptotic path-way is conserved among these species; however, themechanisms that control the regulation of caspases arenot,as exemplified by the limited insights from studiesin fruitf li es.At present,we do not yet have a compre-hensive understanding of the conserved and divergentmechanisms of caspase regulation across species.

conserved in Diap1,which indicates a different mode ofinhibit ion.Biochemical analyses showed that Diap1directly inhibits the catalytic activity of Drice through itsBIR1 domain,and that this inhibit ion can be counteredeffectively by the RHG proteins61. Interestingly,Diap1binds to and inhibits Drice only after the cleavage of itsN-terminal 20 amino acids61,which could serve as anauto-inhibitory sequence for Diap1 function. Diap1-mediated inhibit ion of Drice involves a highly con-served surface groove on the BIR1 domain61.Crystalstructures of BIR1 bound to the RHG peptides showthat the RHG proteins use their N-terminal IAP-bind-ing motif to bind to the same surface groove61, therebyremoving Diap1-mediated inhibition of Drice.Despitethese biochemical advances,we do not yet understandthe exact molecular mechanism of how Diap1 inhibitsDrice.

Cleavage after residue Asp20 of Dr ice could haveanother impor tant function; that is, to accelerate thedegradation of the full -length Diap1 protein throughtheN-END RULE112.Since Diap1 is readily converted into anN-terminal Drice-inhibiting BIR1 domain and a C-ter-minal Dronc-targeting domain by the catalytic activityof Dronc61, this proposed mechanism might also lead tothe removal of the BIR1 domain.Alternati vely,sinceDrice forms a stable complex with the BIR1 domain of

Diap1(REF.61), the proposed N-end rule could facilitatethe degradation of Drice as well .

The frontiers

Recent investigations of caspase regulation have led to asignificantly improved understanding of the mechanismof activation of effector caspases,the mechanisms of IAP-and p35-mediated inhibit ion of caspases, and themechanisms of removal of IAP-mediated inhibi tion ofcaspases by a diverse fami ly of proteins that share theIAP-binding tetrapeptide moti f. It is important to real-ize that the conserved IAP-binding tetrapeptide motifhas an essential role not only in caspase inhibition,but

N-END RULE

A ubiquitin-dependent pathway

that targets proteins for

degradation through their

destabilizing N-terminal

residues.

Hid

N

Dronc

ba

BIR2 of

Diap1

Phe118Phe5

N

C

CN

Figure 10 | Molecular mechanism of RHG-mediated removal of Dronc suppression by

Diap1.a | M utual exclusion of binding of the fruitflyDrosophila melanogastercaspase-9

homologue Dronc and that of RH G proteins (Reaper, H id, G rim and Sickle), which are

SM AC /DIABLO (direct inhibitor of apoptosis (IA P)-binding protein with low pI) homologues, to the

same conserved surface groove on the BIR2 (baculoviral IAP repeat-2) domain of Diap1. The IAP -

binding motif of RHG-protein Hid (magenta) and the Dronc peptide (blue) are shown. b | A close-up

view of the overlapping binding between Dronc and RHG to the same conserved surface groove

on the BIR 2 domain of D iap1. T his figure was prepared using PyM ol (see the online links box).



R E V I EWS

Drice. More impor tantly,we have li tt le informationon the activation of Dronc,which is thought to be dif-ferent from it s mammalian orthologue, caspase-9.Our past experience does not allow us to predict howDronc is activated in any convincing manner. But it i scertain that the APAF1 homologue Dark has animportant role in this process.

In conclusion, with the exception of ini tiator-caspaseactivation, the molecular mechanisms of caspase regula-tion in mammals have been delineated with an unprece-dented clarity.To gain a comprehensive understandingof caspase regulation, future efforts should be directed atcaspase pathways in fruitfl ies and nematodes.

Although the apoptotic pathway in the nematodehas been genetically characterized1,122,123, the molecularmechanisms by which EGL-1, CED-9, CED-4 andCED-3 operate remain unknown. At present,we haveno knowledge of the molecular mechanisms of howCED-3,the founding member of the apoptotic-caspasefamily, is activated. It remains unclear whether CED-4alone is suffi cient for the activation of CED-3.Nor dowe understand whether CED-4 functi ons as part of aCED-3-containing holoenzyme in a manner that is sim-ilar to the caspase-9-apoptosome,or whether it merelyfacil itates the auto-activation of CED-3. In fruitfl ies,we do not yet understand how Diap1 interactswith

1. Horvitz, H. R. Worms, life, and death (Nobel Lecture).

Chembiochem. 4, 697711 (2003).2. Jacobson, M . D., Weil, M . & Raff, M . C. Programmed cell

death in animal development. Cell88, 347354 (1997).3. Danial, N. N. & Korsmeyer, S. J. Cell death: critical control

points. Cell116, 205219 (2004).

4. Wang, X. The expanding role of mitochondria in apoptosis.

Genes Dev. 15, 29222933 (2001).

5. Abrams, J. M. An emerging blueprint for apoptosis inDrosophila. Trends Cell Biol. 9, 435440 (1999).

6. Thompson, C . B. Apoptosis in the pathogenesis and

treatment of disease. Science267, 14561462 (1995).7. Hanahan, D. & Weinberg, R . A. The hallmarks of cancer.

Cell100, 5770 (2000).8. Yuan, J. & Yankner, B. A . Apoptosis in the nervous system.

Nature407, 802809 (2000).9. G reen, D. R. & Evan, G. I. A matter of life and death. Cancer

Cell1, 1930 (2002).10. Vaux, D. L. & Flavell, R. A. Apoptosis genes and

autoimmunity.Curr. Opin. Immunol. 12, 719724 (2000).11. K err, J. F. F., Wylie, A. H . & C urrie, A. R. Apoptosis: a basic

biological phenomenon with wide-ranging implications in

tissue kinetics. Br. J. Cancer26, 239257 (1972).12. Shi, Y. M echanisms of caspase inhibition and activation

during apoptosis. Mol. Cell9, 459470 (2002).13. Thornberry, N. A. & L azebnik, Y. Caspases: enemies within.

Science281, 13121316 (1998).14. Alnemri, E. S. et al. Human IC E/CED -3 protease

nomenclature. Cell87, 171 (1996).15. Thornberry, N. A. et al. A novel heterodimeric cysteine

protease is required for interleukin-1 processing in

monocytes. Nature356, 768774 (1992).16. C erreti, D. P. et al. M olecular cloning of the interleukin-1

converting enzyme. Science256, 97100 (1992).References 15 and 16 report the identification of thefirst caspase.

17. Yuan, J., Shaham, S., Ledoux, S., Ellis, H. M . & Horvitz, H. R.

The C. eleganscell death geneCed-3encodes a protein

similar to mammalian interleukin-1-converting enzyme.

Cell75, 641652 (1993).Describes the cloning of the first apoptotic caspase,

the nematode protein CED-3.18. Xue, D., Shaham, S. & H orvitz, H. R. TheCaenorhabditis

eleganscell-death protein C ED -3 is a cysteine protease with

substrate specificities similar to those of the human CP P32

protease. Genes Dev. 10, 10731083 (1996).19. K umar, S. & D oumanis, J. The fly caspases.Cell Death

Differ.7, 10391044 (2000).

20. Ahmad, M . et al. Spodo ptera frugiperdacaspase-1, a novelinsect death protease that cleaves the nuclear immunophilin

FK BP 46, is the target of the baculovirus antiapoptotic

protein p35. J. Biol. Chem. 272, 14211424 (1997).21. M adeo, F. et al. A caspase-related protease regulates

apoptosis in yeast. Mol. Cell9, 911917 (2002).22. Budihardjo, I., O liver, H. , Lutter, M ., Luo, X. & Wang, X.

Biochemical pathways of caspase activation during

apoptosis. Annu. Rev. Cell Dev. Biol. 15, 269290 (1999).23. Fesik, S . W. Insights into programmed cell death through

structural biology. Cell103, 273282 (2000).24. Fesik, S. W. & Shi, Y. Controlling caspases.Science294,

14771478 (2001).

25. Shi, Y. A structural view of mitochondria-mediated

apoptosis. Nature Struct. Biol. 8, 394401 (2001).26. Earnshaw, W. C ., M artins, L. M . & Kaufmann, S. H.

M ammalian caspases: structure, activation, substrates, and

functions during apoptosis. Annu. Rev. Biochem . 68,383424 (1999).

27. Adams, J. M . & Cory, S. Apoptosomes: engines for caspase

activation. Curr. Opin. Cell Biol. 14, 715720 (2002).28. Shi, Y . Apoptosome: the cellular engine for the activation of

caspase-9. Structure10, 285288 (2002).29. Li, P . et al. Cytochromecand dATP-dependent formation of

Apaf-1/caspase-9 complex initiates an apoptotic protease

cascade. Cell91, 479489 (1997).Reports the requirement of cytochromec and dATP

for the activation of the initiator caspase, caspase-9.30. Jiang, X. & Wang, X. Cytochrome cpromotes caspase-9

activation by inducing nucleotide binding to Apaf-1. J. Biol.

Chem. 275, 3119931203 (2000).31. Rodriguez, J. & Lazebnik, Y. C aspase-9 and Apaf-1 form an

active holoenzyme. Genes Dev. 13, 31793184 (1999).Formulates the concept of a holoenzyme for theinitiator caspase, caspase-9.

32. Zou, H., L i, Y., Liu, X. & Wang, X. An APAF-1cytochromec

multimeric complex is a functional apoptosome that

activates procaspase-9. J. Biol. Chem. 274, 1154911556(1999).

33. Saleh, A., S rinivasula, S. M ., A charya, S., Fishel, R. &

Alnemri, E. S. C ytochrome cand dATP-mediated

oligomerization of Apaf-1 is a prerequisite for procaspase-9

activation. J. Biol. Chem. 274, 1794117945 (1999).34. Nagata, S . Fas ligand-induced apoptosis. Annu. Rev. Genet.

33, 2955 (1999).35. Peter, M . E. & Krammer, P. H. The CD95(APO -1/Fas) DISC

and beyond. Cell Death Differ. 10, 2635 (2003).

36. Luo, X., Budihardjo, I., Zou, H. , Slaughter, C . & Wang, X.Bid, a B cl2 interacting protein, mediates cytochrome c

release from mitochondria in response to activation of cell

surface death receptors. Cell94, 481490 (1998).37. Li, H., Zhu, H., Xu, C. & Yuan, J. Cleavage of BID by

caspase-8 mediates the mitochondrial damage in the fas

pathway of apoptosis. Cell94, 491501 (1998).38. Yang, X., C hang, H. Y. & Baltimore, D . Essential role of

CED-4 oligomerization in C ED-3 activation and apoptosis.

Science281, 13551357 (1998).39. Hengartner, M . O. & Horvitz, H. R. C. eleganscell survival

gene ced-9encodes a functional homolog of the

mammalian proto-oncogene bcl-2. Cell76, 665676

(1994).

40. Conradt, B. & Horvitz, H. R. TheC. elegansprotein EG L-1 is

required for programmed cell death and interacts with the

Bcl-2-like protein C ED-9.Cell93, 519529 (1998).41. Cory, S. & Adams, J. M . The Bcl2 family: regulators of the

cellular life-or-death switch. Nature Rev. Cancer2, 647656(2002).

42. Rodriguez, A . et al. Dark is a Drosophilahomologue ofApaf-1/CED-4 and functions in an evolutionarily conserved

death pathway. Nature Cell Biol. 1, 272279 (1999).43. Kanuka, H . et al. C ontrol of the cell death pathway by

Dapaf-1, a DrosophilaApaf-1/CED-4-related caspase

activator. Mol. Cell4, 757769 (1999).44. Zhou, L., Song, Z., T ittel, J. & Steller, H. H AC -1, a

Drosophilahomolog of Apaf-1 and C ED-4 functions in

developmental and radiation-induced apoptosis. Mol. Cell4,745755 (1999).

45. Rodriguez, A., Chen, P., Oliver, H. & A brams, J. M.

Unrestrained caspase-dependent cell death caused by loss

of Diap1 function requires theDrosophilaApaf-1 homolog,

Dark. EMBO J. 21, 21892197 (2002).46. Walker, N. P. et al. C rystal structure of the cysteine protease

interleukin-1-converting enzyme: a (p20/p10)2

homodimer.

Cell78, 343352 (1994).47. Wilson, K . P . et al. Structure and mechanism of interleukin-1

converting enzyme. Nature370, 270275 (1994).

References 46 and 47 report the first crystal structureof a caspase.

48. Schweizer, A. , Briand, C. & G rutter, M . G . Crystal structure

of caspase-2, apical initiator of the intrinsic apoptotic

pathway. J. Biol. Chem. 278, 4244142447 (2003).49. Rotonda, J. et al. The three-dimensional structure of

apopain/CP P32, a key mediator of apoptosis. Nature

Struct. Biol. 3, 619625 (1996).

50. M ittl, P. R . et al. Structure of recombinant human CP P32 incomplex with the tetrapeptide acetyl-Asp-Val-Ala-Asp

fluoromethyl ketone. J. Biol. Chem. 272, 65396547 (1997).51. Wei, Y . et al. The structures of caspases-1, -3, -7 and -8

reveal the basis for substrate and inhibitor selectivity. Chem.

Biol. 7, 423432 (2000).52. Watt, W. et al. The atomic-resolution structure of human

caspase-8, a key activator of apoptosis. Structure7,11351143 (1999).

53. Blanchard, H . et al. The three dimersional structure of

caspase-8: an initiator enzyme in apoptosis. Structure7,11251133 (1999).

54. Renatus, M ., Stennicke, H. R. , Scott, F. L., Liddington, R. C.

& Salvesen, G . S . D imer formation drives the activation of

the cell death protease caspase 9. Proc. Natl Acad. Sci.

USA 98, 1425014255 (2001).55. Forsyth, C. M ., Lemongello, D., LaC ount, D. J., Friesen, P. D.

& Fisher, A . J. Crystal structure of an invertebrate caspase.

J. Biol. Chem. 279, 70017008 (2004).56. C hai, J. et al. C rystal structure of a procaspase-7 zymogen:

mechanisms of activation and substrate binding. Cell107,399407 (2001).

57. Riedl, S. J. et al. Structural basis for the activation of human

procaspase-7. Proc. Natl Acad. Sci. USA 98, 1479014795(2001).

References 56 and 57 describe the activationmechanism for an effector caspase, caspase-7.

58. Srinivasula, S. M . et al. G eneration of constitutively active

recombinant caspase-3 and-6 by rearrangement of their

subunits. J. Biol. Chem. 273, 1010710111 (1998).59. Wang, S., Hawkins, C ., Yoo, S., M uller, H.-A. & Hay, B. T he

Drosophilacaspase inhibitor DIAP 1 is essential for cell

survival and is negatively regulated by HID . Cell98, 453463(1999).

60. Hawkins, C. J. et al. TheDrosophilacaspase DR O NC

cleaves following glutamate or aspartate and is regulated by

DIAP1, HID, and GRIM . J. Biol. Chem. 275, 2708427093(2000).

61. Yan, N . et al. M olecular mechanisms of DrICE inhibition by

DIAP 1 and removal of inhibition by Reaper, H id, and G rim.

Nature Struct. Mol. Biol. 11, 420428 (2004).62. Thornberry, N. A. et al. A combinatorial approach definesspecificities of members of the caspase family and

granzyme B. Functional relationships established for key

mediators of apoptosis. J. Biol. Chem. 272, 1790717911(1997).

63. Ni, C. -Z., Li, C., Wu, J. C., Spada, A. P. & Ely, K. R .

C onformational restrictions in the active site of unliganded

human caspase-3. J. Mol. Recognit. 16, 121124 (2003).64. Deveraux, Q. L . & R eed, J. C . IAP family proteins

suppressors of apoptosis. Genes Dev. 13, 239252 (1999).65. Salvesen, G . S. & Duckett, C. S. IA P proteins: blocking the

road to deaths door. Nature Rev. Mol. Cell Biol. 3, 401410(2002).

66. M iller, L. K . An exegesis of IAP s: salvation and surprises

from BIR motifs. Trends Cell Biol. 9, 323328 (1999).67. Hay, B . A. U nderstanding IAP function and regulation: a

view from Drosophila. Cell Death Differ. 7, 10451056(2000).


11/11

NATURE REVI EWS | MOLECULAR CELL BIOLOGY VOLUME 5 | NOVEMBER 2004 | 9 0 7

R E V I EWS

68. M aier, J. K . et al. The neuronal apoptosis inhibitory protein is

a direct inhibitor of caspases 3 and 7. J. Neurosci. 22,20352043 (2002).

69. C hai, J. et al. M olecular mechanism of Reaper/Grim/Hid-

mediated suppression of D IAP 1-dependent Dronc

ubiquitination. Nature Struct. Biol. 10, 892898 (2003).Describes the mechanism by which the RHG proteinsantagonize the Diap1-mediated suppression of Droncin fruitflies.

70. Sun, C . et al. NM R structure and mutagenesis of the

inhibitor-of-apoptosis protein XIAP. Nature401, 818822

(1999).71. Hinds, M . G ., N orton, R. S ., Vaux, D. L. & Day, C . L. Solution

structure of a baculoviral inhibitor of apoptosis (IAP ) repeat.

Nature Struct. Biol. 6, 648651 (1999).

72. Ambrosini, G., Adida, C. & Altieri, D. C. A novel anti-

apoptosis gene, survivin, expressed in cancer and

lymphoma.Nature Med. 3, 917921 (1997).73. Ashhab, Y., Alian, A., P olliack, A., P anet, A. & Yehuda, D. B.

Two splicing variants of a new inhibitor of apoptosis gene

with different biological properties and tissue distribution

pattern. FEBS Lett. 495, 5660 (2001).74. K asof, G . M . & G omes, B. C. Livin, a novel inhibitor of

apoptosis protein family member. J. Biol. Chem. 276,32383246 (2001).

75. Vucic, D., Stennicke, H. R ., P isabarro, M. T., Salvesen, G. S.

& D ixit, V. M . M L-IAP, a novel inhibitor of apoptosis that is

preferentially expressed in human melanomas. Curr. Biol.

10, 13591366 (2000).76. Riedl, S. J. et al. Structural basis for the inhibition of

caspase-3 by XIAP. Cell104, 791800 (2001).

77. C hai, J. et al. Structural basis of caspase-7 inhibiton byXIAP. Cell104, 769780 (2001).78. Huang, Y . et al. Structural basis of caspase inhibition by

XIA P: d ifferential roles of the linker versus the BIR domain.

Cell104, 781790 (2001).References 7678 report the mechanism of effector-caspase inhibition by XIAP.

79. Sun, C . et al. NM R structure and mutagenesis of the third

BIR domain of the inhibitor of apoptosis protein XIA P. J. Biol.

Chem. 275, 3377733781 (2000).80. Shiozaki, E. N . et al. M echanism of XIAP -mediated inhibition

of caspase-9. Mol. Cell11, 519527 (2003).Describes the mechanism of caspase-9 inhibition byXIAP.

81. Srinivasula, S. M . et al. A conserved XIAP-interaction motif in

caspase-9 and Smac/DIA BL O mediates opposing effects

on caspase activity and apoptosis. Nature409, 112116(2001).

Reports the mechanism by which SMAC/DIABLO

antagonizes XIAP-mediated inhibition of caspase-9.82. Bump, N. J. et al. Inhibition of ICE fam ily proteases by

baculovirus antiapoptotic protein p35. Science269,18851888 (1995).

83. Zhou, Q . et al. Interaction of the baculovirus anti-apoptotic

protein p35 with caspases. Specificity, kinetics, and

characterization of the caspase/p35 complex. Biochemistry

37, 1075710765 (1998).84. Riedl, S. J., R enatus, M ., Snipas, S. J. & Salvesen, G. S.

M echanism-based inactivation of caspases by the

apoptotic suppressor p35. Biochemistry40, 1327413280(2001).

85. Xu, G . et al. Covalent inhibition revealed by the crystal

structure of the caspase-8/p35 complex. Nature410,494497 (2001).

Describes the mechanism of caspase inhibition by theviral protein p35.

86. Zoog, S . J., Schiller, J. J., Wetter, J. A., C hejanovsky, N. &

Friesen, P. D. Baculovirus apoptotic suppressor P 49 is a

substrate inhibitor of initiator caspases resistant to P35

in vivo. EMBO J. 21, 51305140 (2002).

87. Pei, Z. et al. Characterization of the apoptosis suppressor

protein P49 from theSpodoptera littoralis

nucleopolyhedrovirus.J. Biol. Chem. 277, 4867748684(2002).

88. Jabbour, A. M . et al. The p35 relative, p49, inhibits

mammalian and Drosophilacaspases including DRO NC

and protects against apoptosis. Cell Death Differ. 9,13111320 (2002).

89. Huntington, J. A. , R ead, R. J. & C arrell, R. W. Structure of a

serpinprotease complex shows inhibition by deformation.

Nature407, 923926 (2000).

90. Shi, Y. A conserved tetrapeptide motif: potentiating

apoptosis through IA P-binding. Cell Death Differ. 9, 9395(2002).

91. Du, C. , Fang, M ., Li, Y. & Wang, X. Smac, a mitochondrial

protein that promotes cytochrome c-dependent caspase

activation during apoptosis. Cell102, 3342 (2000).

92. Verhagen, A. M . et al. Identification of DIAB LO , amammalian protein that promotes apoptosis by binding to

and antagonizing IA P proteins. Cell102, 4353 (2000).93. Wu, G . et al. Structural basis of IAP recognition by

Smac/DIABLO. Nature408, 10081012 (2000).

94. Liu, Z. et al. Structural basis for binding of Smac/DIA BL O to

the XIAP BIR 3 domain. Nature408, 10041008 (2000).References 93 and 94 report the molecularmechanism of IAP recognition by SMAC/DIABLO.

95. Chai, J. et al. Structural and biochemical basis of apoptotic

activation by Smac/DIA BLO . Nature406, 855862 (2000).Reports that the seven N-terminal amino acids ofmature SMAC/DIABLO is sufficient to counter XIAP-mediated inhibition of caspase-9.

96. Suzuki, Y . et al. A serine protease, H trA2, is released from

the mitochondria and interacts with XIAP, inducing cell

death. Mol. Cell8, 613621 (2001).

97. Hegde, R . et al. Identification of Om i/HtrA2 as a

mitochondrial apoptotic serine protease that disrupts

inhibitor of apoptosis proteincaspase interaction. J. Biol.

Chem. 277, 432438 (2002).98. M artin, L. M . et al. The serine protease O mi/HtrA2 regulatesapoptosis by binding XIA P through a R eaper-like motif.

J. Biol. Chem. 277, 439444 (2002).99. van Loo, G . et al. The serine protease O mi/H trA2 is released

from mitochondria during apoptosis. O mi interacts with

caspase-inhibitor XIAP and induces enhanced caspase

activity. Cell Death Differ. 9, 2026 (2002).

100. Verhagen, A . M . et al. HtrA2 promotes cell death through its

serine protease activity and its ability to antagonize inhibitor

of apoptosis proteins. J. Biol. Chem. 277, 445454 (2002).101. Li, W. et al. Structural insights into the pro-apoptotic

function of mitochondrial serine protease HtrA2/O mi. Nature

Struct. Biol. 9, 436441 (2002).102. Tenev, T., Zachariou, A ., W ilson, R ., P aul, A . & M eier, P.

Jafrac2 is an IAP antagonist that promotes cell death by

liberating Dronc from DIAP 1. EMBO J. 21, 51185129(2002).

103. Wu, J.-W., C ocina, A. E., Chai, J., H ay, B. A . & S hi, Y.

Structural analysis of a functional DIA P1 fragment bound to

G rim and Hid peptides. Mol. Cell8, 95104 (2001).104. M eier, P., S ilke, J., Leevers, S. J. & Evan, G. I. The

Drosophilacaspase DR O NC is regulated by DIA P1. EMBO J.

19, 598611 (2000).105. Wilson, R . et al. The DIAP 1 RING finger mediates

ubiquitination of Dronc and is indispensable for regulating

apoptosis. Nature Cell Biol. 4, 445450 (2002).106. M uro, I., H ay, B. A. & Clem, R. J. TheDrosophilaDIAP1

protein is required to prevent accumulation of a continuously

generated, processed form of the apical caspase DR O NC .

J. Biol. Chem. 277, 4964449650 (2002).107. G oyal, L., M cCall, K ., A gapite, J., H artwieg, E. & Steller, H .

Induction of apoptosis byDrosophilareaper, hid and grim

through inhibition of IAP function. EMBO J. 19, 589597(2000).

108. Yoo, S. J. et al. Hid, R pr and Grim negatively regulate DIA P1

levels through distinct mechanisms. Nature Cell Biol. 4,416424 (2002).

109. Ryoo, H. D ., B ergmann, A. , G onen, H ., C iechanover, A. &

Steller, H. Regulation ofDrosophilaIAP1 degradation and

apoptosis by reaper and ubcD1. Nature Cell Biol. 4,

432438 (2002).

110. H olley, C. L ., O lson, M . R., Colon-Ramos, D. A . & K ornbluth, S.

Reaper eliminates IAP proteins through stimulated IA P

degradation and generalized translational inhibition. Nature

Cell Biol. 4, 439444 (2002).

111. Huang, Y., R ich, R. L., M yszka, D. G . & Wu, H. R equirement

of both the second and third BIR domains for the relief of

X-linked inhibitor of apoptosis protein (XIA P)-mediated

caspase inhibition by Smac. J. Biol. Chem. 278,4951749522 (2003).

112. D itzel, M . et al. Degradation of DIA P1 by the N-end rule

pathway is essential for regulating apoptosis. Nature Cell

Biol. 5, 467473 (2003).113. Shi, Y. C aspase activation: revisiting the induced proximity

model. Cell117, 855858 (2004).114. Boatright, K. M . et al. A unified model for apical caspase

activation. Mol. Cell11, 529541 (2003).115. Donepudi, M ., M ac Sweeney, A. , Briand, C. & G rutter, M . G .Insights into the regulatory mechanism for caspase-8

activation. Mol. Cell11, 543549 (2003).116. Salvesen, G . S . & Dixit, V. M . C aspase activation: the

induced-proximity model. Proc. Natl Acad. Sci. USA 96,1096410967 (1999).

117. M uzio, M ., Stockwell, B. R ., Stennicke, H. R ., S alvesen, G. S .

& Dixit, V. M . A n induced proximity model for caspase-8

activation. J. Biol. Chem. 273, 29262930 (1998).118. Yang, X., Chang, H . Y. & Baltimore, D . A utoproteolytic

activation of pro-caspases by oligomerization. Mol. Cell1,319325 (1998).

119. Srinivasula, S. M ., Ahmad, M ., Fernandes-Alnemri, T. &

Alnemri, E. S. Autoactivation of procaspase-9 by

Apaf-1-mediated oligomerization. Mol. Cell1, 949957(1998).

120. M acCorkle, R. A., Freeman, K. W. & Spencer, D. M .

Synthetic activation of caspases: artificial death switches.

Proc. Natl Acad. Sci. USA 95, 36553660 (1998).

121. Boatright, K . M . & Salvesen, G . S. M echanisms of caspaseactivation. Curr. Opin. Cell Biol. 15, 725731 (2003).122. Horvitz, H . R . G enetic control of programmed cell death in

the nematodeCaenorhabd itis elegans. Cancer Res. 59,17011706 (1999).

123. Hengartner, M . O . P rogrammed cell death in invertebrates.

Curr. Opin. Genet. Dev.6, 3438 (1996).124. K raulis, P. J. M olscript: a program to produce both detailed

and schematic plots of protein structures. J. Appl.

Crystallogr.24, 946950 (1991).125. Nicholls, A ., S harp, K . A. & H onig, B . P rotein folding and

association: insights from the interfacial and thermodynamic

properties of hydrocarbons. Proteins: Struct. Funct. Genet.

11, 281296 (1991).

AcknowledgementsWe would like to thank N . Yan for help with figure preparation and

members of the Shi laboratory for discussion. This research is sup-

ported by the National Institutes of Health.

C ompeting interests statementThe authors declare no competing financial interests.

Online links

DATABASES

The following terms in this article are linked online to:

Entrez: http://www.ncbi.nlm. nih.gov/entrez/query.fcgi

ced-3| ced-4| ced-9| egl-1

Flybase: http://flybase.bio.indiana.edu/

Damm | Dcp1 | D ecay | Diap2 | D redd | Drice | Dronc | Grim | H id |

Reaper | S ickle | Strica | Thread

Swiss-Prot: http://us.expasy.org/sprot/

AIF | A PAF1 | A pollon | BAK | BC L2 | B ID | caspase-2 | caspase-3 |

caspase-6 | caspase-7 | caspase-8 | caspase-9 | caspase-10 |

c-IAP1 | c-IAP 2 | CrmA | cytochrome c| DIABLO | EndoG |

FADD | Fas | FasL | HT RA 2 | ICE | ILP2 | M L-IAP | NA IP | p35 |

XIAP

FURTHER INFORMATION

Nature Reviews poster Apoptosis Pathways and Drug

Targets: http://www.nature.com/reviews/poster/apoptosis

The PyMOL Molecular Graphics System:

http://www.pymol.org

Access to this links box is available online.

caspases & cancer

Documents