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Article 4:
Biochemistry of apoptosis. Michael Hengartner. Nature 407, 770, 2000.
Article 5:
Structure and zymogen activation of caspases. M. Donepudi, M. Grütter. Biophysical
Chemistry 101-102: 145-153, 2002.
insight review articles
Apoptosis — the regulated destruction of a cell — is a complicated process. The decision to die cannot be
taken lightly, and the activity of many genes influence a cell’s likelihood of activating its self-destruction
programme. Once the decision is taken, proper execution of the apoptotic programme requires the
coordinated activation and execution of multiple subprogrammes. Here I review the basic components of the
death machinery, describe how they interact to regulate apoptosis in a coordinated manner, and discuss the
main pathways that are used to activate cell death.
M
ulticellular animals often need to get rid It slices, it dices, and that’s not all!
of cells that are in excess, in the way, or What exactly do the caspases do that is so important for
potentially dangerous. To this end, they apoptosis? Activation of caspases does not result in the
use an active dedicated molecular wholesale degradation of cellular proteins. Rather, caspases
programme. As important as cell division selectively cleave a restricted set of target proteins, usually at
and cell migration, regulated (or programmed) cell death one, or at most a few positions in the primary sequence
allows the organism to tightly control cell numbers and (always after an aspartate residue). In most cases, caspase-
tissue size, and to protect itself from rogue cells that mediated ‘protein surgery’ results in inactivation of the tar-
threaten homeostasis. get protein (Box 1). But caspases can also activate proteins,
Discovered and rediscovered several times by various either directly, by cleaving off a negative regulatory domain,
developmental biologists and cytologists, programmed cell or indirectly, by inactivating a regulatory subunit (Box 1).
death acquired a number of names over the past two cen- Several important caspase substrates have been identified
turies1. The term finally adopted is apoptosis, coined by in recent years. One of the more exciting discoveries has been
Currie and colleagues in 1972 to describe a common type of the elucidation of the mechanism of activation of the nuclease
programmed cell death that the authors repeatedly responsible for the famous nucleosomal ladder. First
observed in various tissues and cell types2. The authors described by Wyllie10, this nuclease cuts the genomic DNA
noticed that these dying cells shared many morphological between nucleosomes, to generate DNA fragments with
features, which were distinct from the features observed in lengths corresponding to multiple integers of approximately
cells undergoing pathological, necrotic cell death, and 180 base pairs. The presence of this DNA ladder has been used
they suggested that these shared morphological features (and abused) extensively as a marker for apoptotic cell death.
might be the result of an underlying common, conserved, In an elegant series of experiments, the groups of Wang
endogenous cell death programme3. and Nagata showed that the DNA ladder nuclease (now
known as caspase-activated DNase, or CAD) pre-exists in
Caspases: the central executioners living cells as an inactive complex with an inhibitory
Most of the morphological changes that were observed by subunit, dubbed ICAD (ref. 11). Activation of CAD occurs
Kerr et al. are caused by a set of cysteine proteases that are by means of caspase-3-mediated cleavage of the inhibitory
activated specifically in apoptotic cells. These death subunit, resulting in the release and activation of the
proteases are homologous to each other, and are part of a catalytic subunit12–14.
large protein family known as the caspases4. Caspases are Caspase-mediated cleavage of specific substrates also
highly conserved through evolution, and can be found from explains several of the other characteristic features of apop-
humans all the way down to insects, nematodes and tosis. For example, cleavage of the nuclear lamins is required
hydra5–7. Over a dozen caspases have been identified in for nuclear shrinking and budding15,16. Loss of overall cell
humans; about two-thirds of these have been suggested to shape is probably caused by the cleavage of cytoskeletal
function in apoptosis7,8. proteins such as fodrin and gelsolin17. Finally, caspase-
All known caspases possess an active-site cysteine, and mediated cleavage of PAK2, a member of the p21-activated
cleave substrates at Asp-Xxx bonds (that is, after aspartic kinase family, seems to mediate the active blebbing
acid residues); a caspase’s distinct substrate specificity is observed in apoptotic cells. Interestingly, in this last case,
determined by the four residues amino-terminal to the caspase cleavage occurs between the negative regulatory
cleavage site9. Caspases have been subdivided into subfami- subunit and the catalytic subunit, and results in a constitu-
lies based on their substrate preference, extent of sequence tive activation of PAK2 (ref. 18).
identity and structural similarities. Close to 100 additional caspase substrates have been
Because they bring about most of the visible changes that reported over the years, and there will certainly be many
characterize apoptotic cell death, caspases can be thought of more7,19. Why are there so many substrates? Perhaps apoptosis
as the central executioners of the apoptotic pathway. Indeed, is just much more complicated that we currently believe.
eliminating caspase activity, either through mutation or the Indeed, several of the key apoptotic subprogrammes, such as
use small pharmacological inhibitors, will slow down or even cell shrinking and the emission of pro-engulfment signals (see
prevent apoptosis7. Thus, blocking caspases can rescue con- review in this issue by Savill and Fadok, pages 784–788), are
demned cells from their apoptotic fate — a fact that has not still poorly understood. Alternatively, it is possible that many
escaped the notice of the pharmaceutical industry (see review of the described caspase substrates are not relevant substrates,
in this issue by Nicholson, pages 810–816). but simply ‘innocent bystanders’ that get caught in the action.
770 © 2000 Macmillan Magazines Ltd NATURE | VOL 407 | 12 OCTOBER 2000 | www.nature.com
insight review articles
Box 1 which are found in the mature enzyme. In all cases examined so far,
Camera, lights, action. Cut! Outcome of caspase activity the mature enzyme is a heterotetramer containing two p20/p10
heterodimers and two active sites7. Although much has been made
Proteolytic cleavage by caspases can lead to diverse results, about the fact that active caspases are dimers containing two active
depending on the nature of the substrate and the exact position of sites, there is no obvious structural reason why this should be so, and
the cleavage site in the primary sequence. The simplest, and it seems quite possible that caspases could exist as active monomers
probably most frequent outcome is loss of biological activity under the right conditions.
(panels a, b in the figure below). Caspase substrates range from Three general mechanisms of caspase activation have been
single polypeptide chain enzymes (for example, polyADP-ribose described so far. Each of them is described briefly below (see also Box 2).
polymerase) to complex macromolecular structures (for example, Processing by an upstream caspase
the lamin network). Limited proteolysis by caspases can also result Most caspases are activated by proteolytic cleavage of the zymogen
in a gain of biological activity (c, d). In some cases (for example, between the p20 and p10 domains, and usually also between the
Bcl-2 or Bcl-xL), the cleaved products antagonize the full-length prodomain and the p20 domain. Interestingly, all these cleavage sites
protein (dominant-negative forms). In other cases, removal of occur at Asp-X sites — candidate caspase substrate sites — suggesting
inhibitory domains or subunits results in increased biological the possibility of autocatalytic activation9. Indeed, the simplest way to
activity (for example, PAK2, Bid and CAD/ICAD). activate a procaspase is to expose it to another, previously activated
caspase molecule (Box 2). This ‘caspase cascade’ strategy of caspase
Monomeric substrates Multiprotein complexes activation is used extensively by cells for the activation of the three
short prodomain caspases, caspase-3, -6 and -7. These three down-
a Inactivation b Disassembly stream effector caspases are considered the workhorses of the caspase
family, and are usually more abundant and active than their long
prodomain cousins.
The caspase cascade is a useful method to amplify and integrate
pro-apoptotic signals, but it cannot explain how the first, most
upstream caspase gets activated. At least two other approaches are
used to get the ball rolling.
Loss of function
Induced proximity
Caspase-8 is the key initiator caspase in the death-receptor pathway
(see review in this issue by Krammer, pages 789–795). Upon ligand
binding, death receptors such as CD95 (Apo-1/Fas) aggregate and
form membrane-bound signalling complexes (Box 3). These com-
+ plexes then recruit, through adapter proteins, several molecules of
procaspase-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) is
c Activation d Release sufficient to allow the various proenzyme molecules to mutually
cleave and activate each other (Box 2). A similar mechanism of action
has 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 many
cases to activate caspases22, it is a rather crude a way to control the fate
Gain of function
Keep your friends close, but keep your enemies closer pro p20 p10
Regulated protein–protein interactions are also key to the under-
standing of a second set of apoptotic regulators, the Bcl-2 family. This
b
family 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 III
+ +
promote cell death.
How do Bcl-2 family members control cell death? Bcl-2 family
members seem to spend most of their time simply trying to block
each other’s next move. Many family members can homodimerize,
but more importantly, pro- and anti-apoptotic members can form
heterodimers34–36. Because each Bcl-2 family member can interact c Apaf-1 Cytochrome c
with several other different members, large numbers of heterodimer Caspase-9 Active caspase
combinations within a cell are possible. To a first approximation,
heterodimerization can simply be thought as resulting in mutual ATP
neutralization 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 are
sensitive to death; cells with an excess of protective family members
are usually resistant.
But Bcl-2 proteins clearly will need to do more than just talk to
each other if they are to influence cell death. What is the ultimate
output from all these interactions? In the nematode Caenorhabditis
elegans, the anti-apoptotic Bcl-2 homologue CED-9 protects cells
from death by directly binding to and sequestering the Apaf-1 homo- activation of caspase-9 in the cytosol25.
logue CED-4 (ref. 37). Although this is an appealing scenario, a simi- Exactly how cytochrome c manages to cross the mitochondrial
lar interaction has been very difficult, if not impossible, to detect in outer membrane is not yet known, but it is clear that the Bcl-2 family
mammals, at least not under the conditions tested so far38–40. Rather, is intimately involved in the regulation of this process. For example,
the key function of Bcl-2 family members seems to be to regulate the addition of pro-apoptotic Bcl-2 family members to isolated
release of pro-apoptotic factors, in particular cytochrome c, from the mitochondria is sufficient to induce cytochrome c release, whereas
mitochondrial intermembrane compartment into the cytosol35,36. overexpression of Bcl-2 family members will prevent it35,36,41.
How do Bcl-2 family members regulate cytochrome c exit? Several
Mitochondria — the forum of death competing hypotheses have been advanced (Box 5); none of them has
The mitochondrion is not only the cell’s powerhouse, it is also its been proven definitively34–36,41. The three basic models are as follows.
arsenal. Mitochondria sequester a potent cocktail of pro-apoptotic Bcl-2 members form channels that facilitate protein transport
proteins. Most prominent among these is cytochrome c, the humble Based on the structural similarity of Bcl-xL to the pore-forming sub-
electron carrier. Work over the past few years has revealed that unit of diphtheria toxin42, it has been suggested that Bcl-2 proteins
cytochrome c is far from innocuous — in addition to its involvement might act by inserting, following a conformational change, into the
in mitochondrial oxidative phosphorylation, the protein is one of the outer mitochondrial membrane, where they could form channels or
components (in addition to the adaptor protein Apaf-1) required for even large holes. Bcl-2 family members indeed can insert into
772 © 2000 Macmillan Magazines Ltd NATURE | VOL 407 | 12 OCTOBER 2000 | www.nature.com
insight review articles
Box 3
The roads to ruin: two major apoptotic pathways in mammalian cells
synthetic lipid bilayers, oligomerize, and form channels with discrete can bind to it and regulate its channel activity43. As the characterized
conductances34. But it unclear whether such channels would ever be pore size of the VDAC channel is too small to allow proteins to pass
big enough for proteins to pass through. through, this model must assume that VDAC undergoes a significant
Bcl-2 members interact with other proteins to form channels conformational change upon binding to Bcl-2 family members.
Bcl-2 family members interact with many proteins34. One possibility Bcl-2 members induce rupture of the outer mitochondrial membrane
is that pro-apoptotic family members recruit other mitochondrial It is possible that the Bcl-2 family members control homeostasis of
outer membrane proteins into forming a large pore channel. A the mitochondria. In this model, apoptotic signals alter mitochondr-
particularly attractive candidate for such a protein is the voltage- ial physiology (for example, ion exchange or oxidative phosphoryla-
dependent anion channel (VDAC), as several Bcl-2 family members tion) such that the organelle swells, resulting in the physical rupture
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insight review articles
of the outer membrane and release of intermembrane proteins into as the inhibitors-of-apoptosis (IAP) proteins5,53. There would be
the cytosol. The need to form a channel large enough for cytochrome little reason for such proteins to exist if they could not influence the
c to pass through is thereby neatly bypassed as proteins can be apoptotic process.
assumed to simply diffuse through the tears in the lipid bilayer. On the basis of the above, it might seem that cells suffer from a
Mitochondrial homeostasis could be influenced directly by the terminal case of indecisiveness when it comes to apoptotic cell death,
Bcl-2 family members (for example, through the proposed intrinsic letting apoptotic signalling go down endless trails but never fully
ion-channel activity mentioned above) or indirectly, through modu- committing (Box 3). But this impression would be wrong. Indeed,
lation of other mitochondrial proteins. The VDAC protein again is a quite to the contrary, the apoptotic pathway contains a number of
prominent candidate for such regulation, as it is a subunit of the amplification steps and positive feedback loops that insure that a cell
mitochondrial permeability transition pore (PTP), a large channel will either fully commit to death or completely abstain from it. For
whose opening results in rapid loss of membrane potential and example, the fact that procaspases are caspase substrates insures rapid
organellar swelling. Opening of the PTP quickly leads to cytochrome and complete conversion of a pool of proenzymes even if only a few
c release and apoptotic cell death, and pharmacological inhibitors of molecules were initially activated8. Similarly, there is likely to be
the PTP can act as potent inhibitors of cytochrome c release, and positive feedback between caspase activation and cytochrome c exit
hence prevent apoptosis44. However, cytochrome c exit can also occur from mitochondria54,55.
in the absence of membrane potential loss41,44, suggesting that the But positive feedback loops do require the presence of buffers
PTP cannot be the sole target of the Bcl-2 family proteins. and/or dampeners, or even the smallest perturbation would eventu-
Cytochrome c exit is an almost universal feature of apoptotic cell ally lead to full activation and apoptotic death of the cell. The IAP
death. However, in some cases, it is a very late event. For example, proteins might well act as such dampeners. It is possible, for example,
apoptosis induced by death receptors often bypasses the mitochon- that IAPs are not meant to protect cells from frontal suicide assaults,
drial pathway45. As might be expected, from the models discussed but rather to squelch spurious spontaneous caspase activation.
above, such deaths are relatively insensitive to protection by Bcl-2 This idea is further supported by the recent identification of a
(ref. 45), and cytochrome c release into the cytosol is likely to be the mammalian IAP inhibitor, known as Smac47 (for second mitochon-
result of caspase activation, rather than its cause. dria-derived activator of caspases) or DIABLO48 (for direct
Cytochrome c is but one of a host of mitochondrial pro-death IAP-binding protein with low pI). As is the case for Reaper, Hid and
denizens. Also present in mitochondria and released upon induction Grim in Drosophila (ref. 56, and see review in this issue by Meier et al.,
of apoptosis are AIF (a flavoprotein with potent but relatively pages 796–801), Smac/DIABLO binds to IAP family members and
mysterious apoptotic activity46), Smac/DIABLO47,48, and several pro- neutralizes their anti-apoptotic activity. Most interestingly,
caspases, including procaspase-2, -3 and -9 (ref. 44). Release of Smac/DIABLO is normally a mitochondrial protein, but it is released
multiple death-promoting molecules might be necessary to insure into the cytosol in cells induced to die, presumably following the
swift and certain death — part of the plan to insure that activation of same exit route as cytochrome c.
the apoptotic cascade is a one-way proposition. Thus, if a cell is committed to apoptotic death such that it releases
its mitochondrial contents, then Smac/DIABLO will sequester the
Apoptotic antidotes and anti-antidotes IAP proteins and insure that they do not attempt to stop the
Is release of pro-death factors from mitochondria really the point of programme in its tracks. By analogy, anti-apoptotic Bcl-2 family
no return? Several lines of evidence suggest that cells can occasional- members can be thought of as buffers that minimize accidental
ly still be rescued at this stage — at least for a while. First, pharmaco- release of mitochondrial contents. Several other buffer zones
logical inhibitors of caspases will often (but not always) rescue cells probably exist, waiting to be discovered.
from apoptosis49,50. Second, caspase-3- and caspase-9-knockout
mice show reduced neuronal apoptosis during development and a Would apoptosis, by any other name, be as sweet a death?
significant defect in apoptosis following insult7,51,52. Third, Is caspase activation the defining feature of apoptotic cell death? As
mammals (as well as the fruitfly Drosophila and some viruses) I mentioned at the beginning of this review, most if not all of the
carry a family of genes that encode potent caspase inhibitors, known morphological features used to initially describe apoptotic cell
Box 4
Gatekeepers 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 BH4 BH3 BH1 BH2 TM
Bcl-2 family is comprised of well over a dozen proteins, which have Group I Bcl-2
been classified into three functional groups35,36. Members of the first
group, such as Bcl-2 and Bcl-xL, are characterized by four short, Group II Bax
conserved Bcl-2 homology (BH) domains (BH1–BH4). They also
possess a C-terminal hydrophobic tail, which localizes the proteins Bid
Group III
to the outer surface of mitochondria — and occasionally of the Bik
endoplasmic reticulum — with the bulk of the protein facing the
cytosol. The key feature of group I members is that they all possess
anti-apoptotic activity, and protect cells from death. In contrast, group II consists of Bcl-2 family members with pro-apoptotic activity. Members
of this group, which includes Bax and Bak, have a similar overall structure to group I proteins, containing the hydrophobic tail and all but the
most N-terminal, BH4 domain35,36. Structure/function studies suggest that anti- versus pro-apoptotic activity is determined by relatively large
regions of the protein, including two large a-helices that have been proposed to participate in membrane insertion (see text). Group III consists
of a large and diverse collection of proteins whose only common feature is the presence of the ~12–16-amino-acid BH3 domain35. Although
some members of group III, including Bid, are indeed divergent homologues of Bcl-2 and Bax (refs 70, 71), others share little sequence or
structural similarity with group I and II, suggesting that the BH3 domain in these proteins has arisen through convergent evolution41. Classification
of such proteins as Bcl-2 family members is thus more a matter of convenience than a statement of presumed evolutionary relationship.
774 © 2000 Macmillan Magazines Ltd NATURE | VOL 407 | 12 OCTOBER 2000 | www.nature.com
insight review articles
Box 5
Getting 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.
K+ Cl –
VDAC
Cytoplasm ANT
Na +
ATP,ADP
K+
Na +
Cl –
Cyt c
Bcl-2
family member
death are caspase-dependent. But the apoptotic programme is There is much watching and much deducing left to do. Cell death will
much more than just caspases, and in many cell types, activation of continue to be a lively field for the foreseeable future. ■
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776 © 2000 Macmillan Magazines Ltd NATURE | VOL 407 | 12 OCTOBER 2000 | www.nature.com
Biophysical Chemistry 101 – 102 (2002) 145–153
Abstract
Apoptosis is primarily executed by active caspases, which are derived from the inactive zymogens. Structural and
biochemical 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 biochemical
studies of procaspase-9 and -8 have provided insight into the process of procaspase activation. The mechanism of
zymogen activation requires limited proteolysis as for many other proteases. In addition, self-activation through
oligomerization has been demonstrated for the initiator caspases-8, -9 and -10. These studies provide a structural
mechanism for caspase activation, substrateyinhibitor binding, and contribute to the understanding of the biological
role of caspases in the processes of apoptosis.
䊚 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 acid proteases) play an essential role at various
stages of the apoptotic process w4–6x. The highly
Programmed cell death (apoptosis) is an essen- regulated apoptotic process involves an intricate
tial mechanism in the development and homeosta- cascade of events (Fig. 1a). Currently two major
sis of multicellular organisms to eliminate extrinsic pathways are known, which involve api-
unwanted cells w1,2x. Dys-regulated cell death is cal caspases; one of the extrinsic pathways relies
implicated in a growing number of clinical disor- on a cell surface stimulus. Here, the death signal
ders. Excessive apoptosis can lead to ischemic is transmitted through binding of an extracellular
damage and neurodegenerative diseases whereas death ligand such as tumor necrosis factor (TNF)
conditions such as cancer or autoimmune diseases to its cognate receptor, the TNF receptor. For both
result from insufficient apoptosis w3x. ligands and receptors alike, a number of different
Whilst controlled cell death pathways still need homologous proteins are already known, which act
to be fully investigated, biochemical and genetic on cells that must die during tissue development,
have established that caspases (cysteine aspartatic immunological development, and in response to
viral infection. The death receptors transmit signals
*Corresponding author. Tel.: q41-1-635-5580; fax: q41-1-
to the interior of the cells, where the apical
635-6834. proteases of the extrinsic pathway, caspases-2, -8
E-mail address: gruetter@bioc.unizh.ch (M.G. Grutter).
¨ and -10 are recruited w7x. The other pathway occurs
0301-4622/02/$ - see front matter 䊚 2002 Elsevier Science B.V. All rights reserved.
PII: S 0 3 0 1 - 4 6 2 2 Ž 0 2 . 0 0 1 5 1 - 5
146 ¨
M. Donepudi, M.G. Grutter / Biophysical Chemistry 101 – 102 (2002) 145–153
Fig. 1.
¨
M. Donepudi, M.G. Grutter / Biophysical Chemistry 101 – 102 (2002) 145–153 147
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 cell
surface. 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 are
typical of programmed cell death. Irreparable genomic damage caused by mutagens, pharmaceuticals, or ionizing radiation, activates
the intrinsic pathway in which cytochrome c is released from the mitochondria. This event triggers the formation of a complex
between procaspase-9, Apaf-1, and cytochrome c. 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 Ca2q . This leads to the activation of executioner
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 activation
process of caspases. Caspases are synthesized as single chain precursors. Activation proceeds by cleavage of the N-terminal domain
at Asp119, Asp296, and Asp316 (all caspase-1 numbering convention) leading to a large, a, and a small, b, subunit. The activity
and 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 dimer
in which one ayb unit that harbors the active site is related by a twofold axis with a second unit to form the active (ayb)2 dimer.
148 ¨
M. Donepudi, M.G. Grutter / Biophysical Chemistry 101 – 102 (2002) 145–153
tural and functional research related to caspases caspases-1, -4, -5, -11, -12, -13) w4x. The recent
within the last year. The recently published struc- procaspase-7 structures and caspase-9 structure,
tures of procaspase-7 and studies regarding acti- along with biochemical data, provide insights into
vation of procaspase-9 will specifically be the molecular mechanism of caspase activation
reviewed. These experiments provide a preliminary w28,29x.
picture of zymogen activation in caspases and
further our understanding of the biological role of 3. Structure and activation mechanism of pro-
these proteolytic enzymes in programmed cell caspase-7
death.
The structures of procaspase-7 (a caspase zymo-
2. Zymogen activation gen), and an active caspase-7 without ligand,
display significant structural differences in the
Caspases are produced as inactive zymogens, active site cleft region w28x. As in the bound
which are activated by specific proteolytic cleav- caspase-7, the zymogen is composed of two cata-
age. Initiator caspases activate downstream caspas- lytic domains. Each domain contains a central six-
es-3, -6 and -7. However, the activation process stranded b-sheet and five a-helices (Fig. 3). Four
for apical proteases is difficult to rationalize. Pro- surface loops emerge from the core structure to
caspase-8 possesses limited catalytic activity, form the potential active site. Comparison of the
which is sufficient to allow intramolecular proc- loop positions between the zymogen and the active
essing of zymogen molecules at high concentra- enzyme shows large structural differences (Fig. 3)
tions w21,22x. The high concentration is achieved w28,29x.
by zymogen clustering at the cytosolic part of the The procaspase-7 zymogen is a single continu-
death receptor forming the death inducing signal- ous polypeptide chain. It possesses an intersubunit
ling complex (DISC) (Fig. 1a) w23,24x. Activation linker (called L2), which forms a loop between
is mediated by like–like protein interactions the large and the small subunits of the mature
through adaptor proteins w22x. Activation of cas- caspase. This intersubunit segment contains the
pase-9 occurs in a similar manner by the formation cleavage site (Ile195–Gln196–Ala197–Asp198)
of a complex between caspase-9, apoptotic prote- for the activation of procaspase-7. Part of this loop
ase activating factor-1 (Apaf-1) and cytochrome (residues 190–203) was not visible in the struc-
c: the apoptosome w25x. The adaptor protein, Apaf- ture, due to flexibility in the active site region,
1, acts as a cofactor, increasing the proteolytic which is probably required for binding to the active
activity by orders of magnitude w26x (Fig. 1a). site of an activator caspase (Fig. 3). In the crystal
The activation of proteases, generally, proceeds structure, the N-terminus of the small subunit in
by limited proteolysis and removal of an N- one heterodimer traverses the dimeric interface,
terminal peptide or an entire N-terminal domain and is oriented towards the C-terminus of the large
(Fig. 1b). Caspase zymogens possess an N-termi- subunit. This indicates that the heterodimer of
nal prodomain, and a linker peptide within the procaspase-7 is built from a single polypeptide
protease domain, which are cleaved to render an chain.
active caspase w27x. Internal cleavage sites are A second procaspase-7 structure was published
consistent with the ability to auto-activate or to towards the end of last year w29x describing similar
activate other caspases as part of an amplification findings as in the earlier structure. Continuous
cascade. The sequences of the prodomains vary electron density for the intersubunit linkers was
considerably among caspase family members. not observed, however, the direction which the C-
There are small N-terminal peptides (e.g. caspases- terminus of the large subunit follows supports the
3, -6, -7) and large N-terminal domains that are idea that two monomers are brought together in
involved in recruitment andyor activation (e.g. close association, rather than monomer interdigi-
caspases-2, -8, -9, -10 and the cytokine processing tation. Unlike in the active caspase structures,
¨
M. Donepudi, M.G. Grutter / Biophysical Chemistry 101 – 102 (2002) 145–153 149
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) w19x
were superimposed in O w33x. For clarity, the structure of procaspase-7 has been shown in full and only loop regions from the other
two structures have been shown, to highlight the differences. Labels written in parentheses represent alternative nomenclature, which
agrees with caspase-1 nomenclature w14,34x. This figure was produced in SETOR w32x.
where the chain extends straight on after Cys 285, 5. Substrate-induced fit
the zymogen chain turns 908, and travels upwards
toward the activation loop. Linkers in both struc- A structure of an unbound active caspase was
tures insert themselves into the central cavity and also determined as mentioned above w28x. Surpris-
the dimer interface but in an asymmetric manner ingly, a few notable structural differences do exist
(Fig. 3) w28,29x. between bound active caspase-7 and active caspase
alone. The active site cleft is more defined than in
4. The loop bundle procaspase-7, but less defined than in an inhibited
caspase-7, indicating that active caspase-7 adopts
The loop bundle, termed by Chai et al. w28x, an active site conformation, which is intermediary
comprises L2 (intersubunit linker), L4 (Loop-3), between the two extremes. L3 (activation loop)
and L29 (symmetry-related intersubunit linker) and L4 (L3, caspase-1 nomenclature) are in similar
(Fig. 3) and is present in all structures of active positions as that of an inhibited caspase. However,
caspases. Conversely, the procaspase-7 structures L29 is still in a procaspase conformation; it is still
both have disseminated loop bundles; the required flipped 1808 and points downwards towards the
structural arrangement, which favours catalysis, is N-terminal side of L29. Therefore, the loop bundle
not present. L2, which in active caspases extends is not yet fully assembled in unbound, active
outwards to the bulk solvent, is turned 908 such caspase-7, and binding of a substrateyinhibitor is
that the catalytic cysteine is completely turned the catalyst for achieving a correctly assembled
away from the active site w29,28x. L4, which active caspase (Fig. 3) w28x. The flipping of L29
normally lines one side of the active site, now cannot occur prior to intersubunit cleavage in
turns by 608 away from the active site. The C- procaspase-7, thus deeming procaspase-7 as inca-
terminus of L29 in procaspase-7 is actually pable of substrateyinhibitor interaction.
observed as the N-terminus of the small subunit,
in active caspases. This loop flips 1808 to point 6. Procaspase-9
downwards towards its active site rather than
upwards into the bulk solvent to interact with L2 Renatus et al. w20x have shown that caspase-9
and L4 like in the active caspases (Fig. 3). exists primarily as a monomer, under normal phys-
150 ¨
M. Donepudi, M.G. Grutter / Biophysical Chemistry 101 – 102 (2002) 145–153
Fig. 5. A schematic representation of the events leading up to caspase activation. A monomeric procaspase molecule dimerizes; this
event is concentration-dependent for the initiator caspases. Once dimerized, proteolytic processing occurs, which causes some
structural 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- tioner caspases-7, -3 and -6, which all have linker
gesting that cleavage is necessary for optimal peptides that are comparable in length. The situa-
substrateyinhibitor binding, and that dimerization tion in initiator caspases such as caspase-2, -8, -9
precedes cleavage (unpublished results). A model and -10 is somewhat different. At least for caspase-
of procaspase-8 shows that the intersubunit linker 9, its activation seems to be governed by proteo-
could contribute to the sub-optimal catalytic lytic cleavage as well as a monomer–dimer
arrangements in the active site, thus preventing equilibrium. These caspases are activated by a
optimal substrateyinhibitor binding (Fig. 5, general proximity-induced mechanism. In vivo, caspases
caspase activation scheme). are brought together in supramolecular complexes,
such as the DISC for caspase-8, and the apopto-
9. Conclusions some for caspase-9, via their N-terminal domains.
In vitro, the proximity can be obtained by the high
The recent structural and biochemical investi- concentration of the enzyme, e.g. as used in
gations of caspase-7 and -9 provide a more detailed crystallization.
picture of the activation mechanism of caspases. Although all aspects of substrate specificity are
The structures of caspase-7 and procaspase-7 not yet clearly elucidated for the caspases, a
reveal that the active site cleft in procaspase-7 is plethora of structural information already exists.
deformed and is occluded by a linker peptide We have just begun to understand the molecular
between the two domains that form the active site processes involved in activating this family of
after the enzyme is activated. This represents a enzymes. What remains to be determined and
new mechanism of zymogen activation in prote- discovered are the subtleties associated with each
ases. The mechanism might hold true for execu- caspase’s activation mechanism. Do the N-terminal
152 ¨
M. Donepudi, M.G. Grutter / Biophysical Chemistry 101 – 102 (2002) 145–153
prodomains play a role in keeping the caspases w14x N.P. Walker, R.V. Talanian, K.D. Brady, L.C. Dang, et
dormant? Three-dimensional X-ray crystallograph- al., Crystal structure of the cysteine protease interleukin-
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process are needed to answer these questions. al., The three-dimensional structure of apopainyCPP32,
a key mediator of apoptosis, Nat. Struct. Biol. 3 (7)
(1996) 619–625.
Acknowledgments w16x P.R. Mittl, S. Di Marco, J.F. Krebs, X. Bai, et al.,
Structure of recombinant human CPP32 in complex
The financial support by the Swiss National with the tetrapeptide acetyl–Asp–Val–Ala–Asp fluoro-
Science foundation (grant number 3100- methyl ketone, J. Biol. Chem. 272 (10) (1997)
6539–6547.
053957.98) and by the Baugartenstiftung, 8022 w17x H. Blanchard, L. Kodandapani, P.R. Mittl, S.D. Marco,
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