LESSON 3: BIOCHEMISTRY OF APOPTOSIS -CASPASES

LESSON 3: BIOCHEMISTRY OF APOPTOSIS - CASPASES

(M. Van de Casteele)

• Proteases in signal transduction: irreversibility

• Caspases: classification and function

• Enzymatic mechanism of proteolysis

• Caspase substrates

• Recruitment domains (CARD, DD, DED, pyrin): structure

• Function of platforms in the activation of caspases

• Structure of active caspases

Article 4:Biochemistry of apoptosis. Michael Hengartner. Nature 407, 770, 2000.

Article 5:Structure and zymogen activation of caspases. M. Donepudi, M. Grütter. BiophysicalChemistry 101-102: 145-153, 2002.

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insight review articles

Multicellular animals often need to get ridof cells that are in excess, in the way, orpotentially dangerous. To this end, theyuse an active dedicated molecularprogramme. As important as cell division

and cell migration, regulated (or programmed) cell deathallows the organism to tightly control cell numbers andtissue size, and to protect itself from rogue cells thatthreaten homeostasis.

Discovered and rediscovered several times by variousdevelopmental biologists and cytologists, programmed celldeath acquired a number of names over the past two cen-turies1. The term finally adopted is apoptosis, coined byCurrie and colleagues in 1972 to describe a common type ofprogrammed cell death that the authors repeatedlyobserved in various tissues and cell types2. The authorsnoticed that these dying cells shared many morphologicalfeatures, which were distinct from the features observed incells undergoing pathological, necrotic cell death, and they suggested that these shared morphological featuresmight be the result of an underlying common, conserved,endogenous cell death programme3.

Caspases: the central executionersMost of the morphological changes that were observed byKerr et al. are caused by a set of cysteine proteases that areactivated specifically in apoptotic cells. These death proteases are homologous to each other, and are part of alarge protein family known as the caspases4. Caspases arehighly conserved through evolution, and can be found fromhumans all the way down to insects, nematodes andhydra5–7. Over a dozen caspases have been identified inhumans; about two-thirds of these have been suggested tofunction in apoptosis7,8.

All known caspases possess an active-site cysteine, andcleave substrates at Asp-Xxx bonds (that is, after asparticacid residues); a caspase’s distinct substrate specificity isdetermined by the four residues amino-terminal to thecleavage site9. Caspases have been subdivided into subfami-lies based on their substrate preference, extent of sequenceidentity and structural similarities.

Because they bring about most of the visible changes thatcharacterize apoptotic cell death, caspases can be thought ofas the central executioners of the apoptotic pathway. Indeed,eliminating caspase activity, either through mutation or theuse small pharmacological inhibitors, will slow down or evenprevent apoptosis7. Thus, blocking caspases can rescue con-demned cells from their apoptotic fate — a fact that has notescaped the notice of the pharmaceutical industry (see reviewin this issue by Nicholson, pages 810–816).

It slices, it dices, and that’s not all!What exactly do the caspases do that is so important forapoptosis? Activation of caspases does not result in thewholesale degradation of cellular proteins. Rather, caspasesselectively cleave a restricted set of target proteins, usually atone, or at most a few positions in the primary sequence(always after an aspartate residue). In most cases, caspase-mediated ‘protein surgery’ results in inactivation of the tar-get protein (Box 1). But caspases can also activate proteins,either directly, by cleaving off a negative regulatory domain,or indirectly, by inactivating a regulatory subunit (Box 1).

Several important caspase substrates have been identifiedin recent years. One of the more exciting discoveries has beenthe elucidation of the mechanism of activation of the nucleaseresponsible for the famous nucleosomal ladder. Firstdescribed by Wyllie10, this nuclease cuts the genomic DNAbetween nucleosomes, to generate DNA fragments withlengths corresponding to multiple integers of approximately180 base pairs. The presence of this DNA ladder has been used(and abused) extensively as a marker for apoptotic cell death.

In an elegant series of experiments, the groups of Wangand Nagata showed that the DNA ladder nuclease (nowknown as caspase-activated DNase, or CAD) pre-exists inliving cells as an inactive complex with an inhibitory subunit, dubbed ICAD (ref. 11). Activation of CAD occursby means of caspase-3-mediated cleavage of the inhibitorysubunit, resulting in the release and activation of the catalytic subunit12–14.

Caspase-mediated cleavage of specific substrates alsoexplains several of the other characteristic features of apop-tosis. For example, cleavage of the nuclear lamins is requiredfor nuclear shrinking and budding15,16. Loss of overall cellshape is probably caused by the cleavage of cytoskeletal proteins such as fodrin and gelsolin17. Finally, caspase-mediated cleavage of PAK2, a member of the p21-activatedkinase family, seems to mediate the active blebbingobserved in apoptotic cells. Interestingly, in this last case,caspase cleavage occurs between the negative regulatorysubunit and the catalytic subunit, and results in a constitu-tive activation of PAK2 (ref. 18).

Close to 100 additional caspase substrates have beenreported over the years, and there will certainly be manymore7,19. Why are there so many substrates? Perhaps apoptosisis just much more complicated that we currently believe.Indeed, several of the key apoptotic subprogrammes, such ascell shrinking and the emission of pro-engulfment signals (seereview in this issue by Savill and Fadok, pages 784–788), arestill poorly understood. Alternatively, it is possible that manyof the described caspase substrates are not relevant substrates,but simply ‘innocent bystanders’ that get caught in the action.

The biochemistry of apoptosisMichael O. Hengartner

Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, New York 11724, USA (e-mail: [email protected])

Apoptosis — the regulated destruction of a cell — is a complicated process. The decision to die cannot betaken lightly, and the activity of many genes influence a cell’s likelihood of activating its self-destructionprogramme. Once the decision is taken, proper execution of the apoptotic programme requires thecoordinated activation and execution of multiple subprogrammes. Here I review the basic components of thedeath machinery, describe how they interact to regulate apoptosis in a coordinated manner, and discuss themain pathways that are used to activate cell death.

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According to this line of reasoning, there might be little selection againstthe presence of fortuitous caspase cleavage sites on irrelevant proteins,as the cell is about to stop functioning anyway. Further experimentationmight allow this issue to be resolved.

How to activate a caspaseGiven the great importance of caspases in the apoptotic process, it isreasonable to propose that a proper understanding of apoptosis willrequire us to understand how caspases are activated.

As is true of most proteases, caspases are synthesized as enzymati-cally inert zymogens. These zymogens are composed of threedomains: an N-terminal prodomain, and the p20 and p10 domains,

which are found in the mature enzyme. In all cases examined so far,the mature enzyme is a heterotetramer containing two p20/p10 heterodimers and two active sites7. Although much has been madeabout the fact that active caspases are dimers containing two activesites, there is no obvious structural reason why this should be so, andit seems quite possible that caspases could exist as active monomersunder the right conditions.

Three general mechanisms of caspase activation have beendescribed so far. Each of them is described briefly below (see also Box 2).Processing by an upstream caspaseMost caspases are activated by proteolytic cleavage of the zymogenbetween the p20 and p10 domains, and usually also between theprodomain and the p20 domain. Interestingly, all these cleavage sitesoccur at Asp-X sites — candidate caspase substrate sites — suggestingthe possibility of autocatalytic activation9. Indeed, the simplest way toactivate a procaspase is to expose it to another, previously activatedcaspase molecule (Box 2). This ‘caspase cascade’ strategy of caspaseactivation is used extensively by cells for the activation of the threeshort prodomain caspases, caspase-3, -6 and -7. These three down-stream effector caspases are considered the workhorses of the caspasefamily, and are usually more abundant and active than their longprodomain cousins.

The caspase cascade is a useful method to amplify and integratepro-apoptotic signals, but it cannot explain how the first, mostupstream caspase gets activated. At least two other approaches areused to get the ball rolling. Induced proximityCaspase-8 is the key initiator caspase in the death-receptor pathway(see review in this issue by Krammer, pages 789–795). Upon ligandbinding, death receptors such as CD95 (Apo-1/Fas) aggregate andform membrane-bound signalling complexes (Box 3). These com-plexes then recruit, through adapter proteins, several molecules ofprocaspase-8, resulting in a high local concentration of zymogen.The induced proximity model posits that under these crowded con-ditions, the low intrinsic protease activity of procaspase-8 (ref. 20) issufficient to allow the various proenzyme molecules to mutuallycleave and activate each other (Box 2). A similar mechanism of actionhas been proposed to mediate the activation of several other caspases,including caspase-2 and the nematode caspase CED-3 (ref. 21).Although forced crowding of zymogens clearly is sufficient in manycases to activate caspases22, it is a rather crude a way to control the fateof a cell. Whereas the basic concept is probably correct, additionallevels of regulation surely must exist in vivo to modulate the process.Association with a regulatory subunitThe most complex activation mechanism described so far is the one used by caspase-9. Unlike other caspases, proteolytic processingof procaspase-9 has only a minor effect of the enzyme’s catalyticactivity23,24. Rather, the key requirement for caspase-9 activation is itsassociation with a dedicated protein cofactor, Apaf-1 (Box 2).

Apaf-1 was identified through a biochemical approach as one oftwo proteins that are required for caspase-9 activation (the other beingcytochrome c; see below)25,26. Initially believed to be required only tran-siently, for caspase-9 activation, the Apaf-1/caspase-9 complex is nowthought to actually represent the true active form of caspase-9 (ref. 23).Thus, we must view Apaf-1 not simply as a caspase-9 activator, butrather as an essential regulatory subunit of a caspase-9 holoenzyme.This holoenzyme — often referred to as the apoptosome — is a verylarge complex that might well contain several additional proteins27–29.

In summary, effector caspases are usually activated proteolyticallyby an upstream caspase, whereas initiator caspases are activatedthrough regulated protein–protein interactions. The actual molecularmechanisms mediating initiator caspase activation are still unclear and,most likely, much more complex that currently understood.

Regulated protein–protein interactions are in fact one of the under-lying themes in apoptosis, and whole caspase activation pathways can bedrawn without ever invoking a single enzyme (Box 3). I describe belowsome of the more commonly encountered interaction modules.


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Box 1Camera, lights, action. Cut! Outcome of caspase activity

Proteolytic cleavage by caspases can lead to diverse results,depending on the nature of the substrate and the exact position ofthe cleavage site in the primary sequence. The simplest, andprobably most frequent outcome is loss of biological activity(panels a, b in the figure below). Caspase substrates range fromsingle polypeptide chain enzymes (for example, polyADP-ribosepolymerase) to complex macromolecular structures (for example,the lamin network). Limited proteolysis by caspases can also resultin a gain of biological activity (c, d). In some cases (for example,Bcl-2 or Bcl-xL), the cleaved products antagonize the full-lengthprotein (dominant-negative forms). In other cases, removal ofinhibitory domains or subunits results in increased biologicalactivity (for example, PAK2, Bid and CAD/ICAD).

+

Loss

of f

unct

ion

Gai

n of

func

tion

Monomeric substrates Multiprotein complexes

a b

+

c d

Inactivation

Activation

Disassembly

Release

Active


The handshakes that seal the fateEach of the long-prodomain caspases contains in its prodomain a protein–protein interaction module, which allows it to bind to andassociate with its upstream regulators. Caspase-8 and -10 contain adeath-effector domain (DED), whereas caspase-2 and -9 contain a caspase activation and recruitment domain (CARD). These twodomains share little sequence identity, but fold into very similar three-dimensional structures, consisting of six antiparallel a-helices arrangedin a Greek key configuration30. The same fold is also found in the deathdomain, a third protein interaction module present in several upstreamregulators of apoptosis, such as CD95 and the adapter molecular FADD(ref. 31). It seems likely that the death domain, DED and CARD arederived from a common ancestral domain30.

The structure of the death domain, DED and CARD perfectlysuits their function. The antiparallel helices bundle into a tight core,leaving exposed large surfaces onto which evolution has carvedextended protein–protein interaction domains. The particular faceof the module that is used for interaction varies greatly from one protein to the next31–33. Work so far suggests that the death adaptormodules usually mediate intrafamily interactions (that is, deathdomain/death domain, DED/DED and CARD/CARD). However,structural analyses show that there is enough surface area left ondeath domains, DEDs and CARDs to also interact with other proteins. Indeed, death adaptor modules might well act as integra-tion platforms, binding to several different proteins, which couldmodulate their dimerization and hence caspase activation.

Keep your friends close, but keep your enemies closerRegulated protein–protein interactions are also key to the under-standing of a second set of apoptotic regulators, the Bcl-2 family. Thisfamily has been divided into three groups, based on structural similarities and functional criteria (Box 4). Members of group I possess anti-apoptotic activity, whereas members of groups II and IIIpromote cell death.

How do Bcl-2 family members control cell death? Bcl-2 familymembers seem to spend most of their time simply trying to blockeach other’s next move. Many family members can homodimerize,but more importantly, pro- and anti-apoptotic members can formheterodimers34–36. Because each Bcl-2 family member can interactwith several other different members, large numbers of heterodimercombinations within a cell are possible. To a first approximation, heterodimerization can simply be thought as resulting in mutualneutralization of the bound pro- and anti-apoptotic proteins. Thus,the problem collapses into comparing overall levels of pro- and anti-apoptotic family members: cells with more pro-death proteins aresensitive to death; cells with an excess of protective family membersare usually resistant.

But Bcl-2 proteins clearly will need to do more than just talk toeach other if they are to influence cell death. What is the ultimate output from all these interactions? In the nematode Caenorhabditiselegans, the anti-apoptotic Bcl-2 homologue CED-9 protects cellsfrom death by directly binding to and sequestering the Apaf-1 homo-logue CED-4 (ref. 37). Although this is an appealing scenario, a simi-lar interaction has been very difficult, if not impossible, to detect inmammals, at least not under the conditions tested so far38–40. Rather,the key function of Bcl-2 family members seems to be to regulate therelease of pro-apoptotic factors, in particular cytochrome c, from themitochondrial intermembrane compartment into the cytosol35,36.

Mitochondria — the forum of deathThe mitochondrion is not only the cell’s powerhouse, it is also itsarsenal. Mitochondria sequester a potent cocktail of pro-apoptoticproteins. Most prominent among these is cytochrome c, the humbleelectron carrier. Work over the past few years has revealed thatcytochrome c is far from innocuous — in addition to its involvementin mitochondrial oxidative phosphorylation, the protein is one of thecomponents (in addition to the adaptor protein Apaf-1) required for

activation of caspase-9 in the cytosol25.Exactly how cytochrome c manages to cross the mitochondrial

outer membrane is not yet known, but it is clear that the Bcl-2 familyis intimately involved in the regulation of this process. For example,addition of pro-apoptotic Bcl-2 family members to isolated mitochondria is sufficient to induce cytochrome c release, whereasoverexpression of Bcl-2 family members will prevent it35,36,41.

How do Bcl-2 family members regulate cytochrome c exit? Severalcompeting hypotheses have been advanced (Box 5); none of them hasbeen proven definitively34–36,41. The three basic models are as follows.Bcl-2 members form channels that facilitate protein transportBased on the structural similarity of Bcl-xL to the pore-forming sub-unit of diphtheria toxin42, it has been suggested that Bcl-2 proteinsmight act by inserting, following a conformational change, into theouter mitochondrial membrane, where they could form channels oreven large holes. Bcl-2 family members indeed can insert into



Box 2More than one way to skin a cat: mechanisms of caspase activation

Mechanisms of caspase activation include proteolytic cleavage by anupstream caspase (panel a in the figure below), induced proximity (b)and holoenzyme formation (c). Proteolytic cleavage by an upstreamcaspase is straightforward and effective, and is used mostly foractivation of downstream, effector caspases. It is probably also usedfor induction of apoptosis by non-caspase proteases, such asgranzyme B (see review in this issue by Krammer, pages 789–795).In the second mechanism, recruitment or aggregation of multipleprocaspase-8 molecules into close proximity somehow results incross-activation. The actual process is most probably moresophisticated and more tightly regulated than shown in panel b. Inholoenzyme formation, cytochrome c and ATP-dependentoligomerization of Apaf-1 allows recruitment of procaspase-9 intothe apoptosome complex. Activation of caspase-9 is mediated bymeans of conformational change, not proteolysis. Stoichiometry ofthe apoptosome is not known; it is shown in panel c as a hexamersolely for aesthetic reasons.

+ +

pro p10

12

a

b

c Apaf-1

ATP

Cytochrome cCaspase-9 Active caspase

p20


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synthetic lipid bilayers, oligomerize, and form channels with discreteconductances34. But it unclear whether such channels would ever bebig enough for proteins to pass through.Bcl-2 members interact with other proteins to form channelsBcl-2 family members interact with many proteins34. One possibilityis that pro-apoptotic family members recruit other mitochondrialouter membrane proteins into forming a large pore channel. A particularly attractive candidate for such a protein is the voltage-dependent anion channel (VDAC), as several Bcl-2 family members

can bind to it and regulate its channel activity43. As the characterizedpore size of the VDAC channel is too small to allow proteins to passthrough, this model must assume that VDAC undergoes a significantconformational change upon binding to Bcl-2 family members.Bcl-2 members induce rupture of the outer mitochondrial membraneIt is possible that the Bcl-2 family members control homeostasis ofthe mitochondria. In this model, apoptotic signals alter mitochondr-ial physiology (for example, ion exchange or oxidative phosphoryla-tion) such that the organelle swells, resulting in the physical rupture



Box 3The roads to ruin: two major apoptotic pathways in mammalian cells

The death-receptor pathway(left pathway in the figureopposite) is triggered bymembers of the death-receptor superfamily (such asCD95 and tumour necrosisfactor receptor I). Binding ofCD95 ligand to CD95induces receptor clusteringand formation of a death-inducing signalling complex.This complex recruits, via the adaptor molecule FADD (Fas-associated deathdomain protein), multipleprocaspase-8 molecules,resulting in caspase-8activation through inducedproximity (see Box 2).Caspase-8 activation can beblocked by recruitment of thedegenerate caspasehomologue c-FLIP (ref. 61).

The mitochondrialpathway (right) is usedextensively in response toextracellular cues and internalinsults such as DNA damage(see review in this issue byRich et al., pages 777–783).These diverse responsepathways converge onmitochondria, often through the activation of apro-apoptotic member of theBcl-2 family. Unlike Bcl-2,which seems to spend most if not all of its life attached to intracellular membranes, many group II and group III members, including Bax, Bad, Bim and Bid, can shuttle between the cytosol and organelles62–65. The cytosolic forms represent pools of inactive, but battle-ready proteins. Pro-apoptotic signals redirect these proteins to the mitochondria, where the fight for the cell’s fate will take place. Activation of pro-apoptoticmembers can occur through proteolysis, dephosphorylation and probably several other mechanisms35,36.

Pro- and anti-apoptotic Bcl-2 family members meet at the surface of mitochondria, where they compete to regulate cytochrome c exit by amechanism that is still debated (see text). If the pro-apoptotic camp wins, an array of molecules is released from the mitochondrial compartment.Principal among these is cytochrome c, which associates with Apaf-1 and then procaspase-9 (and possibly other proteins) to form theapoptosome. Heat-shock proteins act at multiple steps in the pathway to modulate apoptosis (not shown; see refs. 66, 67).

The death-receptor and mitochondrial pathways converge at the level of caspase-3 activation. Caspase-3 activation and activity is antagonizedby the IAP proteins, which themselves are antagonized by the Smac/DIABLO protein released from mitochondria. Downstream of caspase-3, theapoptotic programme branches into a multitude of subprogrammes, the sum of which results in the ordered dismantling and removal of the cell.

Cross-talk and integration between the death-receptor and mitochondrial pathways is provided by Bid, a pro-apoptotic Bcl-2 family member.Caspase-8-mediated cleavage of Bid greatly increases its pro-death activity, and results in its translocation to mitochondria, where it promotescytochrome c exit. Note that under most conditions, this cross-talk is minimal, and the two pathways operate largely independently of eachother62,68.

Clearly, additional death-inducing pathways must exist, as developmental apoptosis is by and large normal in mice defective in the caspase-8and caspase-9 pathways7,52.

DNA damage

Bid

Truncated bid

Procaspase-8

Procaspase-3

FADD

CD95

CD95L

p53

Bax

Bcl-2

Bcl-xL

Apoptotic substrates

Caspase-3

IAPs

Apoptosome

Procaspase-9

Apaf-1

Caspase-8

AIF

Smac/DIABLO

Cytochrome c

c-FLIP


of the outer membrane and release of intermembrane proteins intothe cytosol. The need to form a channel large enough for cytochromec to pass through is thereby neatly bypassed as proteins can beassumed to simply diffuse through the tears in the lipid bilayer.

Mitochondrial homeostasis could be influenced directly by theBcl-2 family members (for example, through the proposed intrinsicion-channel activity mentioned above) or indirectly, through modu-lation of other mitochondrial proteins. The VDAC protein again is aprominent candidate for such regulation, as it is a subunit of themitochondrial permeability transition pore (PTP), a large channelwhose opening results in rapid loss of membrane potential andorganellar swelling. Opening of the PTP quickly leads to cytochromec release and apoptotic cell death, and pharmacological inhibitors ofthe PTP can act as potent inhibitors of cytochrome c release, andhence prevent apoptosis44. However, cytochrome c exit can also occurin the absence of membrane potential loss41,44, suggesting that thePTP cannot be the sole target of the Bcl-2 family proteins.

Cytochrome c exit is an almost universal feature of apoptotic celldeath. However, in some cases, it is a very late event. For example,apoptosis induced by death receptors often bypasses the mitochon-drial pathway45. As might be expected, from the models discussedabove, such deaths are relatively insensitive to protection by Bcl-2(ref. 45), and cytochrome c release into the cytosol is likely to be theresult of caspase activation, rather than its cause.

Cytochrome c is but one of a host of mitochondrial pro-deathdenizens. Also present in mitochondria and released upon inductionof apoptosis are AIF (a flavoprotein with potent but relatively mysterious apoptotic activity46), Smac/DIABLO47,48, and several pro-caspases, including procaspase-2, -3 and -9 (ref. 44). Release of multiple death-promoting molecules might be necessary to insureswift and certain death — part of the plan to insure that activation ofthe apoptotic cascade is a one-way proposition.

Apoptotic antidotes and anti-antidotesIs release of pro-death factors from mitochondria really the point ofno return? Several lines of evidence suggest that cells can occasional-ly still be rescued at this stage — at least for a while. First, pharmaco-logical inhibitors of caspases will often (but not always) rescue cellsfrom apoptosis49,50. Second, caspase-3- and caspase-9-knockoutmice show reduced neuronal apoptosis during development and asignificant defect in apoptosis following insult7,51,52. Third, mammals (as well as the fruitfly Drosophila and some viruses) carry a family of genes that encode potent caspase inhibitors, known

as the inhibitors-of-apoptosis (IAP) proteins5,53. There would be little reason for such proteins to exist if they could not influence theapoptotic process.

On the basis of the above, it might seem that cells suffer from a terminal case of indecisiveness when it comes to apoptotic cell death,letting apoptotic signalling go down endless trails but never fully committing (Box 3). But this impression would be wrong. Indeed,quite to the contrary, the apoptotic pathway contains a number ofamplification steps and positive feedback loops that insure that a cellwill either fully commit to death or completely abstain from it. Forexample, the fact that procaspases are caspase substrates insures rapidand complete conversion of a pool of proenzymes even if only a fewmolecules were initially activated8. Similarly, there is likely to be positive feedback between caspase activation and cytochrome c exitfrom mitochondria54,55.

But positive feedback loops do require the presence of buffersand/or dampeners, or even the smallest perturbation would eventu-ally lead to full activation and apoptotic death of the cell. The IAPproteins might well act as such dampeners. It is possible, for example,that IAPs are not meant to protect cells from frontal suicide assaults,but rather to squelch spurious spontaneous caspase activation.

This idea is further supported by the recent identification of amammalian IAP inhibitor, known as Smac47 (for second mitochon-dria-derived activator of caspases) or DIABLO48 (for direct IAP-binding protein with low pI). As is the case for Reaper, Hid andGrim in Drosophila (ref. 56, and see review in this issue by Meier et al.,pages 796–801), Smac/DIABLO binds to IAP family members andneutralizes their anti-apoptotic activity. Most interestingly,Smac/DIABLO is normally a mitochondrial protein, but it is releasedinto the cytosol in cells induced to die, presumably following thesame exit route as cytochrome c.

Thus, if a cell is committed to apoptotic death such that it releasesits mitochondrial contents, then Smac/DIABLO will sequester theIAP proteins and insure that they do not attempt to stop the programme in its tracks. By analogy, anti-apoptotic Bcl-2 familymembers can be thought of as buffers that minimize accidentalrelease of mitochondrial contents. Several other buffer zones probably exist, waiting to be discovered.

Would apoptosis, by any other name, be as sweet a death?Is caspase activation the defining feature of apoptotic cell death? AsI mentioned at the beginning of this review, most if not all of themorphological features used to initially describe apoptotic cell



Box 4Gatekeepers and gatecrashers: Bcl-2 family members

Named after the founding member of the family, which was isolated as a gene involved in B-cell lymphoma (hence the name bcl; ref. 69), the Bcl-2 family is comprised of well over a dozen proteins, which havebeen classified into three functional groups35,36. Members of the firstgroup, such as Bcl-2 and Bcl-xL, are characterized by four short,conserved Bcl-2 homology (BH) domains (BH1–BH4). They alsopossess a C-terminal hydrophobic tail, which localizes the proteins to the outer surface of mitochondria — and occasionally of theendoplasmic reticulum — with the bulk of the protein facing the cytosol. The key feature of group I members is that they all possessanti-apoptotic activity, and protect cells from death. In contrast, group II consists of Bcl-2 family members with pro-apoptotic activity. Membersof this group, which includes Bax and Bak, have a similar overall structure to group I proteins, containing the hydrophobic tail and all but themost N-terminal, BH4 domain35,36. Structure/function studies suggest that anti- versus pro-apoptotic activity is determined by relatively largeregions of the protein, including two large a-helices that have been proposed to participate in membrane insertion (see text). Group III consistsof a large and diverse collection of proteins whose only common feature is the presence of the ~12–16-amino-acid BH3 domain35. Althoughsome members of group III, including Bid, are indeed divergent homologues of Bcl-2 and Bax (refs 70, 71), others share little sequence orstructural similarity with group I and II, suggesting that the BH3 domain in these proteins has arisen through convergent evolution41. Classificationof such proteins as Bcl-2 family members is thus more a matter of convenience than a statement of presumed evolutionary relationship.

Group I

Group II

Group III

BH4 BH3 BH1 BH2 TM

Bcl-2

Bax

BidBik


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death are caspase-dependent. But the apoptotic programme ismuch more than just caspases, and in many cell types, activation ofthe apoptotic programme inevitably leads to death, with or withoutcaspases57. Programmed death of cells in which caspases have beenblocked often bears little morphological similarity to apoptosis,and can even look suprisingly similar to classical necrotic celldeath58,59.

But that is not all. Physiological forms of cell death with non-apoptotic morphologies have been known for many years58,60. Howshall we classify such deaths? Atypical apoptosis? Necrosis? Non-apoptotic programmed cell death? Ideally, our final classification willbe determined not by morphology, but by what molecular pathwaysare activated in the dying cell. This will require the development ofever more sophisticated assays for apoptotic proteins.

ConclusionsThe field of apoptosis research took off as a result of the careful obser-vations and astute deductions of a group of dedicated pathologists.As Yogi Berra said, “You can observe a lot by watching.” Althoughmany of the key apoptotic proteins have been identified, we still aremostly in the dark as to molecular mechanisms of action or activationof these proteins. Events downstream of caspases are murky, andthere are kinds of cell death that have not even been touched yet.

There is much watching and much deducing left to do. Cell death willcontinue to be a lively field for the foreseeable future. ■■

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Box 5Getting out the vote: possible mechanisms of action of Bcl-2 family members

Bcl-2 family members have been suggested to act through many different mechanisms34–36,41. From left to right in the figure below, these include:• Formation of a pore, through which cytochrome c (Cyt c) and other intermembrane proteins can escape.• Heterodimerization between pro- and anti-apoptotic family members. Dimerization is achieved when the BH3 domain of one molecule binds

into a hydrophobic pocket formed by the BH1, BH2 and BH3 domains of another family member72. Because of structural constraints, both homodimers and heterodimers are asymmetric molecules.

• Direct regulation of caspases via adaptor molecules, as has been described in C. elegans. Although the CED-4 homologue Apaf-1 is probably not a Bcl-2 family target, other adaptor proteins, such as BAR (ref. 73), the endoplasmic reticulum-localized protein Bap31 (ref. 74) and Aven (ref. 75), have been described in mammals.

• Interaction with other mitochondrial proteins, such as VDAC and the adenosine nucleotide transporter (ANT), either to generate a pore for cytochrome c exit, or to modulate mitochondrial homeostasis (for example, opening of the PTP).

• Oligomerization to form a weakly selective ion channel.

Procaspase

Adaptor

BaxBcl-2

ATP,ADP

ATP, ADP

VDAC

ANTCytoplasm

Cyt c

K+ Cl –

Na +

K+

Cl –Na +

Bcl-2family member


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AcknowledgementsIt is impossible to circumscribe the field of apoptosis in such a short review. I apologize tomy many colleagues for having failed to cite their seminal papers and/or the critical resultsthat clearly demonstrate their favourite model to be right. Many thanks to Y. Lazebnik forcontributions to Box 3, and to Y.L., S. Lowe and members of the apoptosis community atCold Spring Harbor Laboratory for many stimulating discussions. I dedicate this reviewto W. Hengartner on the occasion of his retirement from active mathematical duty.


776 NATURE | VOL 407 | 12 OCTOBER 2000 | www.nature.com© 2000 Macmillan Magazines Ltd

Biophysical Chemistry 101–102(2002) 145–153

0301-4622/02/$ - see front matter� 2002 Elsevier Science B.V. All rights reserved.PII: S0301-4622Ž02.00151-5

Structure and zymogen activation of caspases

Mrudula Donepudi, Markus G. Grutter*¨

Institute of Biochemistry, University of Zurich, Winterthurerstr. 190, 8057 Zurich, Switzerland¨

Abstract

Apoptosis is primarily executed by active caspases, which are derived from the inactive zymogens. Structural andbiochemical studies of caspases-1, -3, -7, -8 and -9 have greatly enhanced our understanding of the structure, function,and specificity of the active form of these enzymes. Only recently, the structures of procaspase-7 and biochemicalstudies of procaspase-9 and -8 have provided insight into the process of procaspase activation. The mechanism ofzymogen activation requires limited proteolysis as for many other proteases. In addition, self-activation througholigomerization has been demonstrated for the initiator caspases-8, -9 and -10. These studies provide a structuralmechanism for caspase activation, substrateyinhibitor binding, and contribute to the understanding of the biologicalrole of caspases in the processes of apoptosis.� 2002 Elsevier Science B.V. All rights reserved.

Keywords: Caspases; Inhibitor binding; Structure comparison; X-ray structure; Apoptosis; Zymogen activation

1. Structural overview of active caspases

Programmed cell death(apoptosis) is an essen-tial mechanism in the development and homeosta-sis of multicellular organisms to eliminateunwanted cellsw1,2x. Dys-regulated cell death isimplicated in a growing number of clinical disor-ders. Excessive apoptosis can lead to ischemicdamage and neurodegenerative diseases whereasconditions such as cancer or autoimmune diseasesresult from insufficient apoptosisw3x.

Whilst controlled cell death pathways still needto be fully investigated, biochemical and genetichave established that caspases(cysteine aspartatic

*Corresponding author. Tel.:q41-1-635-5580; fax:q41-1-635-6834.

E-mail address: [email protected](M.G. Grutter).¨

acid proteases) play an essential role at variousstages of the apoptotic processw4–6x. The highlyregulated apoptotic process involves an intricatecascade of events(Fig. 1a). Currently two majorextrinsic pathways are known, which involve api-cal caspases; one of the extrinsic pathways relieson a cell surface stimulus. Here, the death signalis transmitted through binding of an extracellulardeath ligand such as tumor necrosis factor(TNF)to its cognate receptor, the TNF receptor. For bothligands and receptors alike, a number of differenthomologous proteins are already known, which acton cells that must die during tissue development,immunological development, and in response toviral infection. The death receptors transmit signalsto the interior of the cells, where the apicalproteases of the extrinsic pathway, caspases-2, -8and -10 are recruitedw7x. The other pathway occurs

146 M. Donepudi, M.G. Grutter / Biophysical Chemistry 101 –102 (2002) 145–153¨

Fig. 1.

147M. Donepudi, M.G. Grutter / Biophysical Chemistry 101 –102 (2002) 145–153¨

Fig. 2. Ribbon diagram of the caspase-fold represented by thecaspase-8–Z-DEVD-CHO complex(1F9E.pdb) w30,31x. Thisfigure was generated using the programSETOR w32x.

Fig. 1. (a) Schematic representation of the known apoptotic pathways, in which apical caspases are involved. In the death receptor-induced extrinsic pathway, the apoptotic signal is initiated by direct ligand-mediated trimerization of death receptors at the cellsurface. This leads to the recruitment of adaptor proteins inside the cell, which is followed by the activation of the initiator caspases-2, -8 or -10. The executioner caspases-3, -6 -7 are cleaved and activated, which leads to the limited proteolysis events that aretypical of programmed cell death. Irreparable genomic damage caused by mutagens, pharmaceuticals, or ionizing radiation, activatesthe intrinsic pathway in which cytochromec is released from the mitochondria. This event triggers the formation of a complexbetween procaspase-9, Apaf-1, and cytochromec. Caspase-9 subsequently activates the downstream caspases-3, -7, and possibly -6. Alternatively, procaspase-12, located in the ER, can be activated in the presence of Ca . This leads to the activation of executioner2q

caspases or to the limited proteolysis of substrates. The cytokine pathway leading to inflammation is activated by caspase-1.Caspases-4, -5, -11 and -14 are also involved in inflammatory processes.(b) Schematic representation of the proteolytic activationprocess of caspases. Caspases are synthesized as single chain precursors. Activation proceeds by cleavage of the N-terminal domainat Asp119, Asp296, and Asp316(all caspase-1 numbering convention) leading to a large,a, and a small,b, subunit. The activityand specificity determining residues, R179, H237, C285 and R341 are brought into the necessary structural arrangement for catalysis.C285 is the catalytic nucleophile, H237 represents the general base. The crystal structures reveal that the active enzyme is a dimerin which oneayb unit that harbors the active site is related by a twofold axis with a second unit to form the active(ayb) dimer.2

as a consequence of cellular stress, and is mediatedby cytochromec in the mitochondrion, which leadsto caspase-9 activationw8x. Recently, a third, stress-induced apoptotic pathway via the endoplasmicreticulum was discovered in murine cells wherecaspase-12 is involvedw9x. All these pathwayslead to the activation of downstream executionercaspases, which activate or inactivate their specificcellular protein targets via limited proteolysis(Fig.1a).

Based on phylogenetic analyses, caspases havebeen classified into two subfamilies: the interleu-kin-1b converting enzyme-(ICE-) and theCae-norhabditis elegans protein-like (CED-3-like)family, for which the prototypes are ICE(caspase-1) and CED-3 w10x. To date, 14 mammaliancaspase sequences have been reported, of which11 are of human origin and 3 of murine origin(reviewed inw4x).

All caspases recognize tetrapeptidic sequences,with caspase-2 being the exception, and have astringent specificity for cleaving C-terminally ofaspartic acid residues(Fig. 1b). Downstream cas-pases are targeted and cleaved in a specific fashion,enabling cellular disassembly, which is character-istic of apoptosisw11,12x.

The global fold, the topology, and the quarter-nary structure of caspases define a new family ofcysteine proteases. Three-dimensional X-ray crys-tallographic structures are available for caspase-1w13,14x, caspase-3w15,16x, caspase-8w17,18x, cas-pase-7w19x, and caspase-9w20x. The caspase foldconsists of a large,a subunit and a small,bsubunit(Fig. 2). Eachayb subunit comprises six

b-strands, five parallel(a, b, c, d, e), and oneantiparallel (f), which form a twistedb-sheetstructure with twoa-helices(H2, H3) on one side,and threea-helices (H1, H4, H5) on the other,running approximately parallel to theb-strands.The active site cavity is located at the C-terminalend of the parallelb-strands within eachayb

heterodimer(Fig. 2). Two heterodimers associateand are related by a twofold axis. The entirequarternary arrangement can be described as an(ayb) tetramer, composed of two symmetry-2

relatedayb heterodimers, each representing onefolding unit (Fig. 2). Substrate recognition occursin the active site cleft formed by loop regions fromboth thea and theb subunit. The cleft recognizesa tetrapeptide N-terminal of the canonical cleavagesite Asp-X(Fig. 2). This review focuses on struc-


tural and functional research related to caspaseswithin the last year. The recently published struc-tures of procaspase-7 and studies regarding acti-vation of procaspase-9 will specifically bereviewed. These experiments provide a preliminarypicture of zymogen activation in caspases andfurther our understanding of the biological role ofthese proteolytic enzymes in programmed celldeath.

2. Zymogen activation

Caspases are produced as inactive zymogens,which are activated by specific proteolytic cleav-age. Initiator caspases activate downstream caspas-es-3, -6 and -7. However, the activation processfor apical proteases is difficult to rationalize. Pro-caspase-8 possesses limited catalytic activity,which is sufficient to allow intramolecular proc-essing of zymogen molecules at high concentra-tions w21,22x. The high concentration is achievedby zymogen clustering at the cytosolic part of thedeath receptor forming the death inducing signal-ling complex(DISC) (Fig. 1a) w23,24x. Activationis mediated by like–like protein interactionsthrough adaptor proteinsw22x. Activation of cas-pase-9 occurs in a similar manner by the formationof a complex between caspase-9, apoptotic prote-ase activating factor-1(Apaf-1) and cytochromec: the apoptosomew25x. The adaptor protein, Apaf-1, acts as a cofactor, increasing the proteolyticactivity by orders of magnitudew26x (Fig. 1a).

The activation of proteases, generally, proceedsby limited proteolysis and removal of an N-terminal peptide or an entire N-terminal domain(Fig. 1b). Caspase zymogens possess an N-termi-nal prodomain, and a linker peptide within theprotease domain, which are cleaved to render anactive caspasew27x. Internal cleavage sites areconsistent with the ability to auto-activate or toactivate other caspases as part of an amplificationcascade. The sequences of the prodomains varyconsiderably among caspase family members.There are small N-terminal peptides(e.g. caspases-3, -6, -7) and large N-terminal domains that areinvolved in recruitment andyor activation (e.g.caspases-2, -8, -9, -10 and the cytokine processing

caspases-1, -4, -5, -11, -12, -13) w4x. The recentprocaspase-7 structures and caspase-9 structure,along with biochemical data, provide insights intothe molecular mechanism of caspase activationw28,29x.

3. Structure and activation mechanism of pro-caspase-7

The structures of procaspase-7(a caspase zymo-gen), and an active caspase-7 without ligand,display significant structural differences in theactive site cleft regionw28x. As in the boundcaspase-7, the zymogen is composed of two cata-lytic domains. Each domain contains a central six-strandedb-sheet and fivea-helices(Fig. 3). Foursurface loops emerge from the core structure toform the potential active site. Comparison of theloop positions between the zymogen and the activeenzyme shows large structural differences(Fig. 3)w28,29x.

The procaspase-7 zymogen is a single continu-ous polypeptide chain. It possesses an intersubunitlinker (called L2), which forms a loop betweenthe large and the small subunits of the maturecaspase. This intersubunit segment contains thecleavage site(Ile195–Gln196–Ala197–Asp198)for the activation of procaspase-7. Part of this loop(residues 190–203) was not visible in the struc-ture, due to flexibility in the active site region,which is probably required for binding to the activesite of an activator caspase(Fig. 3). In the crystalstructure, the N-terminus of the small subunit inone heterodimer traverses the dimeric interface,and is oriented towards the C-terminus of the largesubunit. This indicates that the heterodimer ofprocaspase-7 is built from a single polypeptidechain.

A second procaspase-7 structure was publishedtowards the end of last yearw29x describing similarfindings as in the earlier structure. Continuouselectron density for the intersubunit linkers wasnot observed, however, the direction which the C-terminus of the large subunit follows supports theidea that two monomers are brought together inclose association, rather than monomer interdigi-tation. Unlike in the active caspase structures,


Fig. 3. A superposition of caspase-7 structures are shown here, in stereo, as a ribbon representation. Procaspase-7, shown in yellow(1K88.pdb), wild-type caspase-7, shown in pink(1K86.pdb) w28x, and caspase-7–Ac-DEVD-CHO, shown in cyan(1F1J.pdb) w19xwere superimposed in Ow33x. For clarity, the structure of procaspase-7 has been shown in full and only loop regions from the othertwo structures have been shown, to highlight the differences. Labels written in parentheses represent alternative nomenclature, whichagrees with caspase-1 nomenclaturew14,34x. This figure was produced inSETOR w32x.

where the chain extends straight on after Cys 285,the zymogen chain turns 908, and travels upwardstoward the activation loop. Linkers in both struc-tures insert themselves into the central cavity andthe dimer interface but in an asymmetric manner(Fig. 3) w28,29x.

4. The loop bundle

The loop bundle, termed by Chai et al.w28x,comprises L2(intersubunit linker), L4 (Loop-3),and L29 (symmetry-related intersubunit linker)(Fig. 3) and is present in all structures of activecaspases. Conversely, the procaspase-7 structuresboth have disseminated loop bundles; the requiredstructural arrangement, which favours catalysis, isnot present. L2, which in active caspases extendsoutwards to the bulk solvent, is turned 908 suchthat the catalytic cysteine is completely turnedaway from the active sitew29,28x. L4, whichnormally lines one side of the active site, nowturns by 608 away from the active site. The C-terminus of L29 in procaspase-7 is actuallyobserved as the N-terminus of the small subunit,in active caspases. This loop flips 1808 to pointdownwards towards its active site rather thanupwards into the bulk solvent to interact with L2and L4 like in the active caspases(Fig. 3).

5. Substrate-induced fit

A structure of an unbound active caspase wasalso determined as mentioned abovew28x. Surpris-ingly, a few notable structural differences do existbetween bound active caspase-7 and active caspasealone. The active site cleft is more defined than inprocaspase-7, but less defined than in an inhibitedcaspase-7, indicating that active caspase-7 adoptsan active site conformation, which is intermediarybetween the two extremes. L3(activation loop)and L4(L3, caspase-1 nomenclature) are in similarpositions as that of an inhibited caspase. However,L29 is still in a procaspase conformation; it is stillflipped 1808 and points downwards towards theN-terminal side of L29. Therefore, the loop bundleis not yet fully assembled in unbound, activecaspase-7, and binding of a substrateyinhibitor isthe catalyst for achieving a correctly assembledactive caspase(Fig. 3) w28x. The flipping of L29cannot occur prior to intersubunit cleavage inprocaspase-7, thus deeming procaspase-7 as inca-pable of substrateyinhibitor interaction.

6. Procaspase-9

Renatus et al.w20x have shown that caspase-9exists primarily as a monomer, under normal phys-


Fig. 4. Secondary structure ribbon representation of caspase-9showing the two different active site conformations and resul-tant dimeric asymmetry. Thea subunits are coloured in yellowand theb subunits are coloured in cyan. Nomenclature used inthis figure are in accordance with caspase-1 nomenclature(seeFig. 3 references). This figure was generated inSETOR w32x.

iological concentrations. They used gel filtrationchromatography coupled with activity assays, andglutaraldehyde crosslinking experiments followedby SDS-PAGE, to demonstrate that the majorityof the enzyme exists in an inactive monomericform, which is in equilibrium with an activedimeric form. The crosslinking experiment fol-lowed by SDS-PAGE conducted with caspase-9,in the presence of the inhibitor Z-VAD-FMK,drives the equilibrium towards the dimeric form.At high concentrations, as used in crystallization,the dimeric form is the predominant species. Theidea of a concentration dependent dimer formationpreceding activationw22x is supported by thesefindings. The structure of a caspase-9 inhibitorcomplex was also publishedw20x. The uniquefeature in this caspase structure is the presence oftwo different active site conformations, one for theinhibited form, and the second for the uninhibited(Fig. 4). The active site of the inhibited activecatalytic domain is very similar to other inhibitedcaspase structures. The loops forming the activesite are the shortest in caspase-9, resulting in ashallower binding groove and wider substrate spec-

ificity. Large structural differences are visible inthe region of the catalytic site of the uninhibiteddomain, rendering it inactive. The greatest differ-ences exist in the activation loop(Fig. 4).

7. Caspase-9 dimeric interface

Two features distinguish caspase-9 from othercaspases regarding the dimer interface. Ab-sheetbetween the C-terminus of the large subunit andthe N-terminus of the small subunit is absent whencompared to other caspases. Additionally, a seven-residue loop in the large subunit juts across thedimer interface and is proximal to its symmetry-related loop. These two features could be thereason for the weak dimer interaction in caspase-9. The dimer asymmetry is necessary and inherentto caspase-9, however, it could also be a contrib-uting factor to its instability. Residues Phe-390 andPhe-3909 are symmetry-related residues located onb-strand-8, which is the mainb-strand involvedin the dimeric interface. If the two residues wereto have the equivalent orientations in their respec-tive molecules, they would collide into each other(Fig. 4). Therefore, caspase-9 resolves the problemby having an inactive, asymmetrically related mon-omer w20x.

8. Caspase-9 activation

Renatus et al.w20x show that proteolytic cleav-age between the caspase-9 subunits is not neces-sary for proteolytic activity. Moreover, catalyticactivity is only present in dimeric caspase-9. Basedon their findings, a novel ‘self-priming’ mecha-nism for procaspase-9 activation is proposed. Acti-vation is achieved when two inactive monomerspair up, the priming bulge is inserted into thedimer interface, and the activation loop is properlyorientated in its active conformation. The abovesequence of events permits the alignment of thecatalytic residuesw20x.

It appears that caspase-8 follows a similarsequence of events leading to activation as caspa-se-9. Using gel filtration chromatography, we haveobserved that caspase-8 exists primarily as a mon-omer in solution, but shifts to a dimer when boundto an inhibitor. However, procaspase-8 stays mono-


Fig. 5. A schematic representation of the events leading up to caspase activation. A monomeric procaspase molecule dimerizes; thisevent is concentration-dependent for the initiator caspases. Once dimerized, proteolytic processing occurs, which causes somestructural rearrangements in the active site, thus enabling the cleaved caspase to optimally bind its substrates and inhibitors.

meric even in the presence of an inhibitor, sug-gesting that cleavage is necessary for optimalsubstrateyinhibitor binding, and that dimerizationprecedes cleavage(unpublished results). A modelof procaspase-8 shows that the intersubunit linkercould contribute to the sub-optimal catalyticarrangements in the active site, thus preventingoptimal substrateyinhibitor binding(Fig. 5, generalcaspase activation scheme).

9. Conclusions

The recent structural and biochemical investi-gations of caspase-7 and -9 provide a more detailedpicture of the activation mechanism of caspases.The structures of caspase-7 and procaspase-7reveal that the active site cleft in procaspase-7 isdeformed and is occluded by a linker peptidebetween the two domains that form the active siteafter the enzyme is activated. This represents anew mechanism of zymogen activation in prote-ases. The mechanism might hold true for execu-

tioner caspases-7, -3 and -6, which all have linkerpeptides that are comparable in length. The situa-tion in initiator caspases such as caspase-2, -8, -9and -10 is somewhat different. At least for caspase-9, its activation seems to be governed by proteo-lytic cleavage as well as a monomer–dimerequilibrium. These caspases are activated by aproximity-induced mechanism. In vivo, caspasesare brought together in supramolecular complexes,such as the DISC for caspase-8, and the apopto-some for caspase-9, via their N-terminal domains.In vitro, the proximity can be obtained by the highconcentration of the enzyme, e.g. as used incrystallization.

Although all aspects of substrate specificity arenot yet clearly elucidated for the caspases, aplethora of structural information already exists.We have just begun to understand the molecularprocesses involved in activating this family ofenzymes. What remains to be determined anddiscovered are the subtleties associated with eachcaspase’s activation mechanism. Do the N-terminal


prodomains play a role in keeping the caspasesdormant? Three-dimensional X-ray crystallograph-ic structures of full-length caspases and complexeswith interaction partners involved in the activationprocess are needed to answer these questions.

Acknowledgments

The financial support by the Swiss NationalScience foundation (grant number 3100-053957.98) and by the Baugartenstiftung, 8022Zurich to M.G.G. gratefully acknowledged.¨

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LESSON 3: BIOCHEMISTRY OF APOPTOSIS -CASPASES

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