Biochimica et Biophysica Acta 1813 (2011) 558–563

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Biochimica et Biophysica Acta

j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / b b a m c r

Review

Caspase-8 and Bid: Caught in the act between death receptors and mitochondria☆
Chahrazade Kantari, Henning Walczak ⁎
Tumour Immunology Unit, Division of Immunology and Inflammation, Department of Medicine, Imperial College London, Hammersmith Hospital Campus,
Commonwealth Building Du Cane Road, London W12 0NN, UK

a r t i c l e i n f o a b s t r a c t

Article history: Mitochondria play a central role in maintaining cells alive, but are also important mediators of cell death. The
Received 8 October 2010 main event in mitochondrial signalling and control of apoptosis is the permeabilisation of the outer
Received in revised form 19 January 2011 mitochondrial membrane and the release of pro-apoptotic proteins into the cytosol from the mitochondrial
Accepted 21 January 2011
intermembrane space. With respect to death receptor-mediated apoptosis, the activation of the mitochondrial
Available online 2 February 2011
pathway is required for apoptosis induction in cells which are described as “type II” cells whereas “type I” cells
Keywords:
do not require it. In type I cells, activation of the extrinsic pathway is sufficient to induce apoptosis. This
Caspase-8 review deals with the events that enable cell death in type II cells, i.e., the signals that lead from death receptor
Mitochondria stimulation to permeabilisation of the outer mitochondrial membrane. Caspase-8 and Bid are the known
Bid procurers of the death signal in this part of the apoptotic pathway. Currently many exciting new findings are
Type II cells emerging concerning the regulation of caspase-8 and Bid function and activation. We will take you on a
Death inducing signalling complex journey through these new developments and point out what we consider the major unknowns in this field.
XIAP We end our review on an up-to-date discussion of the determinants of the type I–type II cell distinction. This
article is part of a Special Issue entitled Mitochondria: the deadly organelle.
© 2011 Elsevier B.V. All rights reserved.

1. Current view and emerging concepts in caspase-8 signalling activation at the TRAIL and CD95 DISCs, is neither required for apoptosis
induction in the presence of caspase-8 nor capable of functionally
1.1. Structure substituting caspase-8 in its absence [5].

Caspases belong to a family of highly conserved aspartate-specific
cysteine proteases [1]. In humans, the caspase gene family consists of
11 members that are grouped into two major sub-families, namely the
apoptotic and the inflammatory caspases [2]. The apoptotic caspases
are further subdivided into two sub-groups, initiator and executioner
caspases which together orchestrate the apoptotic cell death
programme. Caspases are synthesised as zymogens which are inactive
precursors consisting of an N-terminal pro-domain and a C-terminal
protease domain. The C-terminal domain is subdivided into the large
(20 kDa) α subunit and the small (10–12 kDa) β subunit. The pro-
domain of caspase-8 consists of two death effector domains (DEDs)
important for its recruitment to the death-inducing signalling complex
(DISC) via a homotypic interaction with FADD. Indeed, caspase-8 is
crucial for triggering apoptosis via death receptors since its recruitment
to and activation at the DISC is the decisive step for the initiation of the
caspase cascade leading to apoptosis [3,4]. There is one other
DED-containing caspase besides caspase-8, caspase-10. Its role in
death receptor signalling was controversial until it was shown that
caspase-10, despite its FADD-dependent but caspase-8-independent
☆ This article is part of a Special Issue entitled Mitochondria: the deadly organelle.
⁎ Corresponding author.
E-mail address: h.walczak@imperial.ac.uk (H. Walczak).
1.2. Recruitment to the DISC and the different models for caspase-8
activation
Two distinct signalling pathways trigger apoptosis: the extrinsic
pathway – also known as the direct, or type I pathway – triggered by
the binding of specific pro-apoptotic ligands belonging to the TNF
superfamily (SF) to a subfamily of TNF receptor (TNFR) SF members
also referred to as “death receptors”, and the intrinsic pathway
activated by cellular damage in which the mitochondrion plays a
central role.
The best-known mode of caspase-8 activation is by triggering of
five of the six death domain (DD)-containing members of the TNFR SF,
also referred to as death receptors. These are CD95 (APO-1, Fas) [6,7],
TRAIL-R1 (DR4) [8,9], TRAIL-R2 (DR5) [10–13], TNFR1 (p55/p60
TNFR) [14,15] and TRAMP (DR3) [16,17]. These receptors are
activated by respective ligands which belong to the TNF SF of
cytokines, namely CD95L (APO-1L/FasL) [18], TRAIL (Apo2L) [19,20],
TNF [21] and TL1A [22]. For DR6, the sixth DD-containing receptor, a
specific amino-terminal cleavage fragment of the β-amyloid precur-
sor protein (APP), N-APP, was recently described as a ligand [23]. No
ligand for DR6 belonging to the TNF SF has been identified, at least so
far, and caspase-8 does not seem to be involved in its signalling [23].
Therefore we will concentrate on the five caspase-8-activating death

0167-4889/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.bbamcr.2011.01.026
 C. Kantari, H. Walczak / Biochimica et Biophysica Acta 1813 (2011) 558–563
receptors. These receptors come in two flavours as they are either
direct recruiters of FADD, as is the case for CD95, TRAIL-R1 and [24]
TRAIL-R2, or of TRADD, as in the case of TNFR1 and TRAMP. Whilst the
primary output of the FADD recruiters is the induction of apoptosis,
the TRADD recruiters primarily activate gene induction that is often
referred to as being pro-inflammatory. However, the gene-inducing
capacity of TRADD-recruiting receptors derived from the primary
complex which forms around the cross-linked receptor (complex I)
regulates the apoptosis-inducing output of a secondary complex
(complex II) which forms subsequently to the receptor-associated
signalling complex in the cytosol [25]. Consequently, under physio-
logical conditions the gene- and cell death-inducing signals are in
balance and it is only when this balance is perturbed that TNF and
most likely also TL1A induce cell death. When signals from complex I
of TRADD-recruiting complexes are blocked, then this complex II
induces apoptosis in a manner quite similar to the events that occur
when the death-inducing signalling complex (DISC) forms upon
cross-linking of the FADD-recruiting receptors by CD95L or TRAIL.
Following ligand binding to their cognate receptors the DD- and
DED-containing adaptor protein FADD (MORT1) is recruited. This
recruitment is due to a homotypic interaction of the FADD DD with
the cytoplasmic DD of CD95, TRAIL-R1 or TRAIL-R2. Following its
recruitment to the activated receptors, FADD in turn recruits
caspase-8, caspase-10, and cFLIP, a caspase-8- and -10-inhibiting
protein, to the TRAIL and CD95 DISCs [5,26]. The recruitment of
caspase-8, caspase-10 and cFLIP requires homotypic interactions
between the DED of FADD and the N-terminal DED of the caspases or
cFLIP [27]. Activation of caspase-8 and -10 occurs via DISC-
recruitment-induced homodimerisation of the caspases which
induces an activating conformational change. But even if recruitment
of caspase-8 to the DISC initiates its activation, full caspase-8
activation requires complex molecular events which are so far not
completely understood.
Currently, a number of models exist on how initiator caspases are
activated and it is not yet clear which one, if any of them, will prevail.
The so-called induced-proximity model was the first model to be
proposed [28]. In this model the recruitment to the receptor complex
by the adaptor protein FADD leads to clustering of initiator procaspases
which in turn results in self-activation via a cross-proteolysis
mechanism. Thereby, initiator caspases are auto-processed when
brought into close proximity of each other. This model has led to the
description of the so-called interdimer processing model of procaspase-
8 activation by Chang et al. in 2003. In this study, the authors highlight
the distinction between procaspase-8 and caspase-8 enzymatic activ-
ities [29]. Indeed, they showed that upon oligomerisation, individual
procaspase-8 molecules associate with each other through their
protease domain to form dimers. Those dimers are enzymatically
active but can only recognise and cleave others dimers resulting in
cross-cleavage. Procaspase-8 activation involves two sequential cleav-
age events, i.e., the separation of the large and the small subunit
followed by the separation of the large subunit from the prodomain,
which lead to the fully active caspase-8 which can cleave caspase-3 and
Bid as its best studied apoptosis substrates.
This first model was re-interpreted into the “proximity-induced
dimerisation” model [30]. This model proposes that dimer formation
drives activation of initiator caspases and the adaptor protein
complexes serve to promote dimerisation by increasing the local
concentrations of initiator caspases. Thus, in this model dimerisation,
and not zymogen cleavage, is crucial for initiator caspase activation,
even though caspase processing stabilises the active dimers. Finally
the “induced conformation model” was proposed by Chao and
collaborators [31] in which the conformation of the active site of the
initiator caspase, attained through direct interaction with the adaptor
protein complex, is crucial for activation. The latter model was based
on studies with caspase-9 activation at the apoptosome, the other
initiator-caspase-activating platform besides the DISC. The active site
559
of caspase-9 is stabilised by the apoptosome which induces activation.
However, caspase-9 can also be activated as a homodimer, as
proposed by the proximity-induced dimerisation model and later its
active site conformation is stabilised by dimer formation and dimer
interactions with the apoptosome. The third possibility is that
caspase-9 is activated as a higher order homo-oligomer and its active
site conformation is stabilised both by the oligomer and the
apoptosome [30,32]. Although these studies were performed with
the initiator caspase-9 at the apoptosome, similar molecular concepts
might be valid for activation of the DISC-associated initiator caspase-8
and -10.
Based on these existing models, a study published by MacFarlane
and colleagues [33] introduced interesting new elements on
caspase-8 activation at the DISC and on the downstream signalling
outcomes. Using a reconstituted functional DISC with purified CD95,
FADD, and procaspase-8, they propose a two-step activation mech-
anism involving both dimerisation and proteolytic cleavage of
procaspase-8. Even more interestingly, they identify this as a key
regulatory step whereby activated death receptor complexes decide
between signal for death or survival. Indeed, they show that the initial
dimerisation of procaspase-8 provides proteases only with a limited
substrate repertoire limited to itself (probably caspase-10) and cFLIP,
and that a secondary proteolytic cleavage is then required to fully
activate caspase-8 so that it can cleave apoptotic substrates like
caspase-3 and Bid.
Recently, Jin et al. proposed that caspase-8 polyubiquitination by
the E3 ligase cullin-3 and its subsequent p62-dependent aggregation
stabilises active caspase-8 and thereby positively regulates apoptosis
[34]. They observed that stimulation of TRAIL-sensitive cells resulted
in polyubiquitination of a small fraction of DISC-associated caspase-8.
They identify cullin-3 as the E3 ubiquitin ligase responsible for this
modification and as a new TRAIL DISC component. In their model,
following its recruitment to the DISC, cullin-3 is able to ubiquitinate
caspase-8 which in turn recruits the ubiquitin-binding protein p62.
This newly formed caspase-8/cullin-3/p62 complex is then thought to
translocate to the cytosol from where it induces apoptosis in a
p62-dependent manner [34–36]. Yet, even though a fraction of
caspase-8 indeed appears to be polyubiquitinated at the DISC (Jin et
al. and our unpublished observation), the proposed molecular
mechanisms for this modification and its functional consequences
have to await confirmation before being fully integrated into our
current model of the molecular events that occur at the DISC. It is
noteworthy that in p62-deficient cancer cells caspase activity and
apoptosis appear to be diminished, yet they are not abrogated. This
suggests that p62 is not essential for extrinsic pathway activation but
required for its full activity, an effect that can turn out to be highly
relevant under physiological conditions since the signalling output of
death receptors is tightly controlled.
2. Bid: the bridge between caspase-8 activated at the DISC and
pro-apoptotic activation of mitochondria
2.1. The role of mitochondria in caspase-8-mediated apoptosis in type II
cells
The mitochondria-dependent (or intrinsic) apoptosis pathway is
activated by diverse stimuli including growth factor withdrawal, DNA
damage, heat shock, UV- or γ-radiation, and chemotherapeutic drugs
[37]. The signalling pathways activated by these stressors culminate in
mitochondrial outer membrane permeabilisation (MOMP), enabling
release of proteins from the mitochondrial intermembrane space [37].
As previously mentioned, in type II cells activation of the mitochondrial
arm of the apoptosis pathway is required for the induction of apoptosis
following a death receptor stimulus. The bridging element between the
two arms of the apoptosis pathway is the caspase-8-mediated cleavage
of the pro-apoptotic Bcl-2 family member Bid [38] (Fig. 1).
560 C. Kantari, H. Walczak / Biochimica et Biophysica Acta 1813 (2011) 558–563

2.2. Cleavage of Bid
Mitochondrial apoptosis is primarily regulated by molecular
interactions between different members of the Bcl-2 family [39,40].
The common feature of members of this family is that they contain
one or more Bcl-2 homology (BH) domains. Based on the number of
regions of sequence homology with Bcl-2 (BH regions 1–4) they
contain, as well as based on their function, members of the Bcl-2
family can be subdivided into pro- and anti-apoptotic members. The
anti-apoptotic proteins of this family are Bcl-2, Bcl-xL, Mcl-1, Bcl-w,
and A1. All of them contain all BH domains, i.e., BH1 to BH4. The pro-
apoptotic Bcl-2 family can be subdivided into two subgroups. The first
subgroup consists of Bax, Bak and Bok, the three multi-domain pro-
apoptotic proteins. The second subgroup, the so-called BH3-only
proteins, so far has about a dozen or so members including proteins
like Bim, Bmf, PUMA, NOXA, Bad and Bid [41]. The current models of
BH3-only protein function at the mitochondria following their
activation are eloquently described in a review by Andrews [73].
Therefore our main focus in the following paragraphs will be on the
events that lead to the activation of Bid, the BH3-only protein that
links death receptor cross-linking to pro-apoptotic events at mito-
chondria. This link is provided by active caspase-8. In its uncleaved
form Bid is generally thought to be inactive as an apoptosis inducer.
Following death receptor stimulation Bid is cleaved by caspase-8. This
cleavage results in the generation of a 15 kDa protein termed
truncated Bid (tBid). tBid is capable of inducing mitochondrial outer
membrane permeabilisation (MOMP) in cells in which the ratio of
pro- and anti-apoptotic Bcl-2 family members allows it to do so.
Thereby, caspase-8-mediated cleavage of Bid into a pro-apoptotically
active, truncated form provides the link between death receptor
stimulation and mitochondrial apoptotic events [38,42].
Bid was first cloned in 1996 as a novel death agonist that
heterodimerises with either agonists (BAX) or antagonists (BCL-2)
[43]. Whilst most studies find that truncation of Bid is crucial for Bid-
induced MOMP [38,41,42,44,45], there are also reports suggesting a
pro-apoptotic role for full-length Bid [43,46]. Interestingly, in a model
for anoikis – i.e., cell death by loss of attachment – endogenous Bid was
shown to translocate to the mitochondria without cleavage [47].
Although the significance of these findings has yet to be assessed in
terms of possible endogenous activation/cleavage events that could

Fig. 1. Schematic representation of the role of mitochondria in caspase-8 signalling. Binding of CD95 or TRAIL to their respective receptors leads to receptor trimerisation and
formation of the death-inducing signalling complex (DISC). The adaptor protein FADD is recruited to the DISC where the death domains (DD) of both proteins interact. Subsequently,
procaspases-8 and -10 are recruited to the protein complex where they interact with FADD via the death effector domains (DEDs). cFLIP can compete with caspase-8 for the binding
to FADD. Therefore, high levels of cFLIP can abrogate caspase-8 activation at the DISC. DISC-activated caspase-8 and -10 trigger a caspase cascade by cleavage of caspase-3. In type I
cells, activation of the extrinsic pathway is sufficient to induce TRAIL- and CD95-induced apoptosis whereas in type II cells, Bid cleavage is required for apoptosis induction by TRAIL
and CD95L. Caspase-8 cleaves Bid into tBid which initiates the mitochondrial apoptosis pathway leading to release of cytochrome c (CytC) and Smac/DIABLO from the mitochondria.
MTCH2/MMP facilitates the translocation of tBid to the outer mitochondrial membrane (OMM). Recently it was proposed that caspase-8 integration into Cardiolipin (CL)-rich
domains of the OMM results in full activation of this caspase which can then directly access and cleave its substrate Bid. After release from mitochondria, CytC, together with Apaf-1
forms the apoptosome, an activation platform for caspase-9. Smac/DIABLO counteracts the inhibitory function of XIAP thereby allowing for full activation of caspase-3 and -9,
ultimately leading to cell death.
 C. Kantari, H. Walczak / Biochimica et Biophysica Acta 1813 (2011) 558–563
follow translocation of full-length Bid to mitochondria, these studies
suggest that cleavage of Bid may not be an absolute requirement for
Bid's pro-apoptotic function. However, in most cases activation by
truncation seems to be required. During apoptosis Bid can be cleaved by
caspase-8 [38], yet several other proteases including granzyme B
[38,48], cathepsins [49,50], and calpains [51], but also caspase-10
[52,53] and caspase-3 [54], have been shown to be capable of cleaving,
and thereby activating, Bid.
The fact that Bid is a caspase-8 substrate and that its cleavage can
be crucial for CD95-induced apoptosis was first reported in 1998
[38,42]. Caspase-8 cleaves Bid at aspartic acid residue 60 (Asp60) [38],
leading to the release of a truncated form containing the carboxy
(C)-terminal part of the protein. The resulting 15 kDa product (p15) is
tBid which has the capacity to rapidly accumulate at mitochondria
and to initiate MOMP. Cleavage of Bid by caspase-8 removes the
N-terminal portion of the protein which, when still linked to the
C-terminal p15 portion inhibits the pro-apoptotic function of the
latter. Accordingly, when Bid is not cleaved it normally does not
accumulate at the outer mitochondrial membrane (OMM). The
inhibitory role of the N-terminal fragment was elegantly demonstrat-
ed by Tan et al. [55]. They showed that the N- and C-terminal
fragments of Bid are able to bind to each other via interaction of the
BH3-B domain of the N-terminal and the BH3 domain of the
C-terminal portion of the protein. Accordingly, mutations in the
BH3-B region of Bid that prevent interaction with the BH3 domain in
its C-terminal portion show apoptosis activity similar to that of tBid
when expressed alone. The authors could thereby conclude that the
amino-terminal portion of Bid contains a region which negatively
regulates the exposure of the BH3-domain and consequently its
binding to the OMM and/or factors included in such membranes. Mc
Donnell et al. reported [56] that this cleavage changes the conforma-
tion of the protein, leading to an exposition of hydrophobic residues,
thereby allowing the insertion of tBid into the membrane and binding
of its BH3 domain to other Bcl-2 family members. In view of these
findings it is tempting to speculate that in cases when full-length Bid
is thought to act pro-apoptotically, the interaction between the BH3-B
and BH3 domains of Bid may be prevented in a manner different from
proteolysis, e.g., by post-translational modifications of Bid or by the
interaction of the BH3-B domain with another factor.
2.3. Translocation of Bid to the mitochondria and initiation of MOMP
In 1999 Gross et al. reported that cytosolic inactive p22 BID is
cleaved by caspases at internal aspartic acid residues to yield a major
p15 fragment and two minor fragments, p13 and p11. The tBid p15
fragment then translocates to mitochondria and inserts into the OMM
[44]. Following binding of Bid to mitochondria Bak and/or Bax
oligomerise in the OMM, and enable cytochrome c release [57,58]. Bid
or tBid alone is unable to permeabilise the OMM without Bax and Bak
[41,59] but as recently reviewed [41], Bid seems to be more closely
related to multi-domain Bcl-2 family members like Bax than to other
BH3-only proteins and therefore its insertion into the OMM is likely to
be more “Bax-like” as well.
However, even though certain aspects of the molecular mechanism
leading to MOMP following tBid translocation are already quite well
established, others are not. As an example, in a recent study the Gross
laboratory identified MTCH2/MIMP as a major player in the events
resulting in MOMP. In this study Zaltsman et al. showed that MTCH2/
MIMP facilitates recruitment of active tBid to mitochondria [45]. They
used different experimental systems including MTCH2/MIMP condi-
tional knockout ES cells and MEFs to demonstrate the importance of
MTCH2/MIMP for both, recruitment of tBid to mitochondria and its pro-
apoptotic role. Finally they establish that this interaction is also relevant
in vivo as they found that MTCH2/MIMP absence in hepatocytes
decreased the sensitivity of MTCH2/MIMP mice to anti-CD95-induced
hepatocellular damage.
561
Hence, this study expands our knowledge about Bid-induced pro-
apoptotic signalling. Yet, at the same time it exemplifies that even
though we now have a fairly good understanding of this process, we
are still far from grasping all the intricate and complex molecular
alterations and interactions that lead to activation of Bid, MOMP and
apoptosis via the mitochondrial pathway following death receptor
stimulation.
2.4. The Cardiolipin enriched “mitosome”: a new mitochondrial
activation platform for caspase-8?
The current view on why mitochondria are important for death-
receptor-mediated apoptosis in type II cells is because their pro-
apoptotic programme is triggered by tBid. The role of caspase-8
activity in the current model is confined to the TRAIL or CD95 DISC
and it is thought to provide the Bid activatory signal from this
platform only. However, in a recent study Gonzalvez et al. challenge
this notion by showing that caspase-8 integration into Cardiolipin
(CL)-rich domains of the OMM results in full activation of this caspase
which can then directly access and cleave its substrate Bid [60].
Several molecules have been suggested in the past to be involved in
caspase-8 translocation to or sequestration at the mitochondria. In
2002, Stegh et al. showed that in MCF7 cells the bifunctional apoptosis
regulator (BAR) binds to, and thereby sequesters and neutralises,
active caspase-8 in a Bcl-2-regulated manner [61]. Others suggested
that, following anti-CD95-induced translocation of caspase-8 to
mitochondria, the caspase-8-binding protein FLICE-associated huge
protein (FLASH) would form a molecular complex with caspase-8,
thereby presumably activating the mitochondrial apoptosis pathway
by regulating caspase-8 activity [62]. Another protein suggested to
play a role in caspase-8 translocation to mitochondria is the
mitochondrial membrane protein Cardif [63,64]. Gonzalvez et al.
are, however, the first to show that the anionic mitochondria-specific
phospho-lipid CL acts as a mitochondria-associated platform that is
actually required for caspase-8 translocation, oligomerisation and
activation after CD95 stimulation, and hence CD95-induced death in
type II cells. The authors first show a correlation between synthesis of
mature CL and sensitivity to CD95-induced apoptosis since cells
obtained Barth syndrome patients and tafazzin-deficient HeLa cells
are resistant to CD95-induced apoptosis. Barth syndrome is a genetic
disorder involving loss of tafazzin expression, a transacylase which is
required for maturation of CL [65]. In CL-deficient cells, resistance is
mediated by a defect in tBid production with the consequence that
cytochrome c and SMAC/DIABLO release from mitochondria and
caspase-3 activation are inhibited. Importantly, the defect was not due
to tBid being incapable of inducing MOMP in these cells as tafazzin-
knockdown cells transiently transfected with a tBid expression vector
were as sensitive to MOMP induction as control cells. As the next
logical step the authors then tested whether caspase-8 was properly
activated in CL-deficient cells and found that this was indeed not the
case; in CL-deficient cells, both caspase-8 translocation to the
mitochondria and its subsequent activation were abrogated. In
summary, Gonzalvez et al. show that Cardiolipin is crucial for
caspase-8 activation and that it allows the insertion of procaspase-
8 into the OMM, where it then homodimerises leading to its
autoactivation. Thus, in type II cells this mechanism is crucial for
cell death induction. Finally the authors suggest the existence of a
“mitosome” in CL-enriched regions of the OMM, bringing together
caspase-8 and its substrate Bid. It is tempting to speculate that
MTCH2/MIMP and its role in Bid recruitment may synergise with
CL-induced mitosome formation to facilitate MOMP.
3. Discrimination between type I and type II cells
Dependency of cells on the mitochondrial pathway to undergo
apoptosis following a death receptor stimulus, defines two cell types,
562 C. Kantari, H. Walczak / Biochimica et Biophysica Acta 1813 (2011) 558–563
namely type I and type II cells. In type II cells, Bid cleavage is required
for apoptosis induction by TRAIL and CD95L whereas type I cells do
not require Bid cleavage and activation of the extrinsic pathway is
sufficient to induce TRAIL- and CD95-induced apoptosis. The
differentiation into type I and type II cells was identified by, and
first thought to be solely due to, differences in formation of the death-
inducing signalling complex (DISC). Indeed it was shown that in type I
cells caspase-8 activation at the DISC was very efficient, resulting in
direct activation of caspase-3 and the caspase cascade. By contrast,
DISC formation in type II cells was found to be weaker and it was
shown that in such cells with weak capability of DISC formation the
mitochondrial arm of the apoptosis pathway was required for CD95 to
kill cells [66,67]. Elegant in vivo proof for the correctness of the
biochemically defined distinction into type I and type II cells was
provided by the finding that Bid-deficient mice survived a single bolus
injection of anti-CD95 antibodies that killed wild-type mice [68].
Although this study provided genetic proof for the correctness of
the type I–type II distinction and the biochemical differences at the
DISC led the way to its discovery, the fact that the molecular
differences in DISC formation were responsible, or even required for
this distinction, were subsequently challenged. An important role in
this challenge was played by the subsequent discovery of the X-linked
inhibitor of apoptosis protein (XIAP) and its role as an inhibitor of
caspase-3, -7 and -9 [24], in combination with the finding that the
second mitochondrial activator of caspases (SMAC), also known as
direct inhibitor of apoptosis-binding protein with low pI (DIABLO),
when released from mitochondria following MOMP can neutralise the
caspase-inhibitory role of XIAP [69]. In a number of biochemical
studies it was shown that the distinction between type I and type II
cells could also be made on the basis of the level of expression of XIAP.
It was found that when XIAP expression was low or absent, cells were
usually of type I and in its presence usually of type II, i.e., in the latter
case Smac/DIABLO release from mitochondria was required to remove
XIAP from effector caspases, thereby enabling their autocatalytic
activation [70]. In 2009, Wilson et al. showed that silencing of cFLIP
which resulted in increased caspase-8 activation was not necessarily
sufficient to convert a type II cell into a type I cell but that combined
inhibition of cFLIP and XIAP enabled Bax-independent apoptosis in
formerly mitochondria-dependent type II colorectal cancer cells [71].
Finally, the role of XIAP in discriminating between type I and type II
was recently also proven genetically in an elegant study by Jost et al.
[72].
Hence, triggering of the DISC leads to caspase-8 activation. Active
caspase-8 cleaves caspase-3 which, in type I cells, leads to cell death
induction. However in type II cells this is blocked by XIAP. However,
active caspase-8 also cleaves Bid and this can then induce MOMP.
MOMP in turn results in the release of pro-apoptotic factors from the
mitochondrial intermembrane space, including cytochrome c and
SMAC/DIABLO. Cytochrome c release triggers apoptosome formation
and activation of caspase-9, whereas release of SMAC/DIABLO results
in neutralisation of XIAP, thereby enabling full maturation and
activation of the effector caspase-3, -7 and -9 and, consequently,
induction of apoptosis. Therefore, cells which express high levels of
XIAP cannot directly activate caspase-3 following DISC-induced
caspase-8 activation which is why in these cells Bid cleavage and
subsequent MOMP are required for apoptosis to occur. Thus, XIAP
expression or lack thereof is an important factor in the classification of
cells as type I versus type II with respect to death receptor-mediated
apoptosis.
This being said, given that the differences in DISC formation
between type I and type II cells exist and that these differences were
in fact the defining element in this distinction, one should not easily
rule out that the DISC-forming capacity of a particular cell type
contributes to its type I versus type II affiliation. It is quite possible
that there are still unidentified differences at the DISC between type I
and type II cells that may co-segregate with presence and/or absence
of XIAP expression. It may therefore be rewarding to investigate the
relationship between these two different modes of pathway control in
more detail and whether crosstalk between them may even exist.
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