Death related ced-3/Nedd2-like protein
The apoptotic signal triggered by ligation of members of the death receptor family is promoted by sequential
activation of caspase zymogens. In a purified system, the initiator caspases-8 and -10
directly process the executioner pro-caspase-3. These rates of activation are of sufficient magnitude to indicate direct processing in vivo. Differentially
processed forms of caspase-3 that accumulate during caspase-3 activation have similar rates of activation, activities, and
specificities. The pattern and rate of caspase-8 induced activation of pro-caspase-3 in cytosolic extracts is the
same as in a purified system. Moreover, immunodepletion of pro-caspase-9, a putative intermediary in the pathway to
activation, is without consequence. Taken together these data demonstrate that the initiator
caspase-8 can directly activate pro-caspase-3 without the requirement for an accelerator. The in vitro data thus
help to deconvolute previous in vivo transfection studies that have debated the role of a direct versus indirect
transmission of the apoptotic signal generated by ligation of death receptors (Stennicke, 1998).
A cytosolic protein has been purified that induces cytochrome c release from mitochondria in
response to caspase-8, the apical caspase activated by cell surface death receptors such as Fas and TNF. Peptide mass fingerprinting identified this protein as Bid, a BH3 domain-containing protein known to interact with both Bcl2 and Bax. Caspase-8 cleaves Bid, and the COOH-terminal part translocates to mitochondria where it triggers cytochrome c release. Immunodepletion of Bid from cell extracts eliminates the cytochrome c releasing activity. The cytochrome c releasing activity of Bid is antagonized by Bcl2. A mutation at the BH3 domain diminishes its cytochrome c releasing activity. Bid, therefore, relays an apoptotic signal from the cell surface to mitochondria (Luo, 1998).
BID, a BH3 domain-containing proapoptotic Bcl2 family member, is a specific proximal
substrate of Casp8 in the Fas apoptotic signaling pathway. While full-length BID is localized in cytosol,
truncated BID (tBID) translocates to mitochondria and thus transduces apoptotic signals from cytoplasmic
membrane to mitochondria. tBID induces first the clustering of mitochondria around the nuclei and release of
cytochrome c independent of caspase activity, and then the loss of mitochondrial membrane potential, cell
shrinkage, and nuclear condensation in a caspase-dependent fashion. Coexpression of BclxL inhibits all the
apoptotic changes induced by tBID. These results indicate that BID is a mediator of mitochondrial damage induced by Casp8 (Li, 1998).
Signaling through the CD95/Fas/APO-1 death receptor plays a critical role in the homeostasis of the immune
system. RICK, a novel protein kinase that regulates CD95-mediated apoptosis has been identified and characterized.
RICK is composed of an N-terminal serine-threonine kinase catalytic domain and a C-terminal region containing
a caspase-recruitment domain. RICK physically interacts with CLARP, a caspase-like molecule known to bind
to Fas-associated protein with death domain (FADD) and caspase-8. Expression of RICK promotes the
activation of caspase-8 and potentiates apoptosis induced by Fas ligand, FADD, CLARP, and caspase-8.
Deletion mutant analysis reveals that both the kinase domain and caspase-recruitment domain are required for
RICK to promote apoptosis. A RICK mutant (in which the lysine of the putative
ATP-binding site at position 38 is replaced by a methionine) functions as an inhibitor of CD95-mediated
apoptosis. Thus, RICK represents a novel kinase that may regulate apoptosis induced by the CD95/Fas receptor
pathway (Inohara, 1998).
Genetic studies of the nematode C. elegans have identified several important
components of the cell death pathway, most notably CED-3, CED-4, and CED-9. CED-4 directly
interacts with the Bcl-2 homolog CED-9 (or the mammalian Bcl-2 family member Bcl-xL) and the
caspase CED-3 (or the mammalian caspases ICE and FLICE). This trimolecular complex of CED-4,
CED-3, and CED-9 is functional in that CED-9 inhibits CED-4 from activating CED-3 and thereby
inhibits apoptosis in heterologous systems. The E1B 19,000-molecular weight protein (E1B 19K) is a
potent apoptosis inhibitor and the adenovirus homolog of Bcl-2-related apoptosis inhibitors. Since
E1B 19K and Bcl-xL have functional similarity, it was hypothesized that E1B 19K interacts with CED-4 and
regulates CED-4-dependent caspase activation. Binding analysis indicates that E1B 19K interacts with
CED-4 in a Saccharomyces cerevisiae two-hybrid assay, in vitro, and in mammalian cell lysates. The
subcellular localization pattern of CED-4 is dramatically changed by E1B 19K, supporting the theory
of a functional interaction between CED-4 and E1B 19K. Whereas expression of CED-4 alone could
not induce cell death, coexpression of CED-4 and FLICE augments cell death induction by FLICE,
which is blocked by expression of E1B 19K. Even though E1B 19K does not prevent FLICE-induced
apoptosis, it does inhibit CED-4-dependent, FLICE-mediated apoptosis, which suggests that CED-4
is required for E1B 19K to block FLICE activation. Thus, E1B 19K functions through interacting
with CED-4, and presumably a mammalian homolog of CED-4, to inhibit caspase activation and
apoptosis (Han, 1998).
The Bcl-2 family member Bcl-xL has often been correlated with apoptosis resistance. In peripheral human T cells, resistance to CD95-mediated apoptosis is characterized by a lack of caspase-8
recruitment to the CD95 death-inducing signaling complex (DISC) and by increased expression of Bcl-xL. This raises the possibility that Bcl-xL directly prevents caspase-8 activation by the
DISC. To test this hypothesis a cell line in which CD95 signaling is inhibited by overexpression of Bcl-xL was
used. In these MCF7-Fas-bcl-xL cells Bcl-xL has no effect on the recruitment of caspase-8 to the DISC. It does not
affect the activity of the DISC nor the generation of the caspase-8 active subunits p18 and p10. In contrast,
cleavage of a typical substrate for caspase-3-like proteases, poly(ADP-ribose) polymerase, is inhibited in
comparison with the control-transfected CD95-sensitive MCF7-Fas cells. To test whether Bcl-xL inhibits
active caspase-8 subunits in the cytoplasm, a number of immunoprecipitation experiments were performed. Using
monoclonal antibodies directed against different domains of caspase-8, anti-Bcl-xL antibodies, or fusion proteins
of glutathione S-transferase with different domains of caspase-8, no evidence for a direct or indirect physical
interaction between caspase-8 and Bcl-xL was found. Moreover, overexpression of Bcl-xL does not inhibit the
activity of the caspase-8 active subunits p18/p10. Therefore, in this cell line that has become resistant to
CD95-induced apoptosis due to overexpression of Bcl-xL, Bcl-xL acts independently and downstream of
caspase-8 (Medema, 1998).
Two cell types have been identified, each using almost exclusively one of two different CD95
(APO-1/Fas) signaling pathways. In type I cells, caspase-8 is activated within seconds and
caspase-3 within 30 min of receptor engagement, whereas in type II cells, cleavage of both caspases
is delayed for approximately 60 min. However, both type I and type II cells show similar kinetics
of CD95-mediated apoptosis and loss of mitochondrial transmembrane potential (DeltaPsim). Upon
CD95 triggering, all mitochondrial apoptogenic activities are blocked by Bcl-2 or Bcl-xL
overexpression in both cell types. However, in type II but not type I cells, overexpression of Bcl-2 or
Bcl-xL blocks caspase-8 and caspase-3 activation as well as apoptosis. In type I cells, induction of
apoptosis is accompanied by activation of large amounts of caspase-8 by the death-inducing
signaling complex (DISC), whereas in type II cells DISC formation is strongly reduced and
activation of caspase-8 and caspase-3 occurs following the loss of DeltaPsim. Overexpression of
caspase-3 in the caspase-3-negative cell line MCF7-Fas, normally resistant to CD95-mediated
apoptosis by overexpression of Bcl-xL, converts these cells into true type I cells in which apoptosis
is no longer inhibited by Bcl-xL. In summary, in the presence of caspase-3 the amount of active
caspase-8 generated at the DISC determines whether a mitochondria-independent apoptosis pathway
is used (type I cells) or not (type II cells) (Scaffidi, 1998).
Apoptosis often involves the release of cytochrome c from mitochondria, leading to caspase activation.
However, in apoptosis mediated by CD95 (Fas/APO-1), caspase-8 (FLICE/MACH/Mch5) is
immediately activated and, in principle, could process other caspases directly. To investigate whether
caspase-8 could also act through mitochondria, active caspase-8 was added to a Xenopus cell-free
system requiring these organelles. Caspase-8 rapidly promotes the apoptotic program, culminating in
fragmentation of chromatin and the nuclear membrane. In extracts devoid of mitochondria, caspase-8
produces DNA degradation, but leaves nuclear membranes intact. Thus, mitochondria are required for
complete engagement of the apoptotic machinery. In the absence of mitochondria, high concentrations
of caspase-8 are required to activate downstream caspases. However, when mitochondria are
present, the effects of low concentrations of caspase-8 are vastly amplified through cytochrome
c-dependent caspase activation. Caspase-8 promotes cytochrome c release indirectly, by cleaving at
least one cytosolic substrate. Bcl-2 blocks apoptosis only at the lowest caspase-8 concentrations,
potentially explaining why CD95-induced apoptosis can often evade inhibition by Bcl-2 (Kuwana, 1998).
Although the molecular mechanisms of TNF signaling have been largely elucidated, the principle that regulates the balance of life and
death is still unknown. The death domain kinase RIP, a key component of the TNF signaling complex, is cleaved
by Caspase-8 in TNF-induced apoptosis. The cleavage site maps to the aspartic acid at position 324 of RIP. The cleavage of RIP results in the blockage of TNF-induced NFkappa-B activation. RIPc, one of the cleavage products,
enhances interaction between TRADD and FADD/MORT1 and increases cell sensitivity to TNF. Most importantly, the Caspase-8 resistant RIP mutants
protect cells against TNF-induced apopotosis. These results suggest that cleavage of RIP is an important process in TNF-induced apoptosis. Furthermore, RIP
cleavage is also detected in other death receptor-mediated apoptosis. Therefore, this study provides a potential mechanism to convert cells from life to death in
death receptor-mediated apoptosis (Lin, 1999).
Previous genetic studies of the nematode C. elegans have identified three important components of cell
death machinery. CED-3 and CED-4 (Drosophila homolog: Apaf-1-related-killer) function to kill cells, whereas CED-9 protects cells from death. Both CED-9
and its mammalian homolog Bcl-xL (a member of the Bcl-2 family of cell death regulators)
interact with and inhibit the function of CED-4. In addition, CED-4 can simultaneously
interact with CED-3 and its mammalian counterparts interleukin-1beta-converting enzyme (ICE) and FLICE. Thus,
CED-4 plays a central role in the cell death pathway, biochemically linking CED-9 and the Bcl-2 family to CED-3
and the ICE family of pro-apoptotic cysteine proteases (Chinnaiyan, 1997).
The CD95 signaling pathway comprises proteins that contain one or two death effector domains (DED), such as
FADD/Mort1 or caspase-8. A novel 37 kDa protein, DEDD, is described that contains an N-terminal DED.
DEDD is highly conserved between human and mouse (98. 7% identity) and is ubiquitously expressed.
Overexpression of DEDD in 293T cells induces weak apoptosis, mainly through its DED by which it interacts
with FADD and caspase-8. Endogenous DEDD is found in the cytoplasm and translocates into the nucleus
upon stimulation of CD95. Immunocytological studies reveal that overexpressed DEDD directly translocates
into the nucleus, where it co-localizes in the nucleolus with UBF, a basal factor required for RNA polymerase I transcription. Consistent with its nuclear localization, DEDD contains two nuclear localization signals and the C-terminal part shares sequence homology with histones. Recombinant DEDD binds to both DNA and
reconstituted mononucleosomes and inhibits transcription in a reconstituted in vitro system. The results suggest that DEDD is a final target of a chain of events by which the CD95-induced apoptotic signal is transferred into the nucleolus to shut off cellular biosynthetic activities (Stegh, 1998).
Adenovirus type 5 encodes a 14.7-kDa protein that protects infected cells from tumor necrosis factor-induced cytolysis by an unknown mechanism. Infection of cells with an adenovirus vector expressing Fas ligand induces rapid apoptosis that is blocked by coinfection with a virus expressing the 14.7-kDa protein. Moreover, FasL promotes the rapid activation of DEVD-specific caspases, and caspase
activation is blocked by coinfection with Adenovirus coding for the 14.7-kDa protein. Cell death induced by the overexpression of Fas ligand, Fas-associated death domain-containing protein (FADD)/MORT1, or FADD-like interleukin-1beta-converting enzyme (FLICE)/caspase-8 in a virus-free system is efficiently blocked by 14.7K expression. Moreover, 14.7K is shown to interact with FLICE. These results support the idea that FLICE is a cellular target for the 14.7-kDa protein (Chen, 1998a).
BH3-only proapoptotic proteins of the Bcl-2 family such as Bad, Bid, Bim, or Bik transduce death stimuli from the cell surface to the central death machinery. Following apoptosis stimulation, these molecules translocate from the cytosol to mitochondria where they bind to membrane-based Bcl-2 family members. Bid plays an essential role in Fas-mediated apoptosis of the so-called type II cells.
In type II cells, such as Jurkat cells or hepatocytes, death-inducing signaling complex (DISC) formation is strongly reduced compared to type I cells in which
activation of large amounts of caspase 8 by the DISC enables direct activation of downstream caspases leading to irreversible cell damage. In type II cells, following cleavage by caspase 8, the C-terminal fragment of Bid translocates to mitochondria and triggers the release of apoptogenic factors, thereby inducing cell death. Bid is phosphorylated by casein kinase I (CKI) and casein kinase II (CKII). Inhibition of CKI and CKII accelerates Fas-mediated apoptosis and Bid cleavage, whereas hyperactivity of the kinases delays apoptosis. When phosphorylated, Bid is insensitive to caspase 8 cleavage in vitro. Moreover, a mutant of Bid that cannot be phosphorylated was found to be more toxic than wild-type Bid. Together, these data indicate that phosphorylation of Bid represents a new mechanism whereby cells control apoptosis (Desagher, 2001).
Fas (APO-1/CD95) is a member of the tumor necrosis factor receptor (TNF-R) family and
induces apoptosis when crosslinked with either Fas ligand or agonistic antibody (Fas antibody). The Fas-Fas
ligand system has an important role in the immune system, where it is involved in the downregulation of immune
responses and the deletion of peripheral autoreactive T lymphocytes. The intracellular domain of Fas interacts
with several proteins including FADD (MORT-1), DAXX, RIP, FAF-1, FAP-1 and Sentrin. The adaptor
protein FADD can, in turn, interact with the cysteine protease caspase-8 (FLICE/MACH/Mch5). In
a genetic screen for essential components of the Fas-mediated apoptotic cascade, a Jurkat T
lymphocyte cell line deficient in caspase-8 has been isolated that was completely resistant to Fas-induced apoptosis.
Complementation of this cell line with wild-type caspase-8 restores Fas-mediated apoptosis. Fas activation of
multiple caspases and of the stress kinase p38 and c-Jun NH2-terminial kinase (JNK) is completely blocked
in the caspase-8-deficient cell line. Furthermore, the cell line is severely deficient in cell death induced by
TNF-alpha and is partially deficient in cell death induced by ultraviolet irradiation, adriamycin and etoposide.
This study provides the first genetic evidence that caspase-8 occupies an essential and apical
position in the Fas signaling pathway and suggests that caspase-8 may participate broadly in multiple apoptotic
pathways (Juo, 1998).
Inhibitor of apoptosis (IAP) gene products play an evolutionarily conserved role in regulating programmed cell
death in diverse species ranging from insects to humans. Human XIAP, cIAP1 and cIAP2 are direct inhibitors
of at least two members of the caspase family of cell death proteases: caspase-3 and caspase-7. The mechanism by which IAPs interfere with activation of caspase-3 and other effector caspases was compared in
cytosolic extracts where caspase activation was initiated by caspase-8, a proximal protease activated by ligation
of TNF-family receptors, or by cytochrome c, which is released from mitochondria into the cytosol during
apoptosis. These studies demonstrate that XIAP, cIAP1 and cIAP2 can prevent the proteolytic processing of
pro-caspases -3, -6 and -7 by blocking the cytochrome c-induced activation of pro-caspase-9. In contrast, these
IAP family proteins do not prevent the caspase-8-induced proteolytic activation of pro-caspase-3; however, they
subsequently inhibit active caspase-3 directly, thus blocking downstream apoptotic events such as further
activation of caspases. These findings demonstrate that IAPs can suppress different apoptotic pathways by
inhibiting distinct caspases and identify pro-caspase-9 as a new target for IAP-mediated inhibition of apoptosis (Deveraux, 1998).
Tumor necrosis factor alpha (TNF-alpha) binding to the TNF receptor (TNFR) potentially initiates
apoptosis and activates the transcription factor nuclear factor kappa B (NF-kappaB), which
suppresses apoptosis by an unknown mechanism. The activation of NF-kappaB blocks
the activation of caspase-8. TRAF1 (TNFR-associated factor 1), TRAF2, and the
inhibitor-of-apoptosis (IAP) proteins c-IAP1 and c-IAP2 have been identified as gene targets of
NF-kappaB transcriptional activity. In cells in which NF-kappaB is inactive, all of these proteins
are required to fully suppress TNF-induced apoptosis, whereas c-IAP1 and c-IAP2 are sufficient
to suppress etoposide-induced apoptosis. Thus, NF-kappaB activates a group of gene products that
function cooperatively at the earliest checkpoint to suppress TNF-alpha-mediated apoptosis and that
function more distally to suppress genotoxic agent-mediated apoptosis (Wang, 1998).
Cells of the monocyte/macrophage lineage play a central role in both innate and acquired immunity of the
host. However, the acquisition of functional competence and the ability to respond to a variety of activating
or modulating signals require maturation and differentiation of circulating monocytes and entails alterations
in both the biochemical and phenotypic profiles of the cells. The process of activation also confers survival
signals essential for the functional integrity of monocytes enabling the cells to remain viable in
microenvironments of immune or inflammatory lesions that are rich in cytotoxic inflammatory mediators
and reactive free-radical species. However, the molecular mechanisms of activation-induced survival
signals in monocytes remain obscure. To define the mechanistic basis of activation-induced resistance to
apoptosis in human monocytes at the molecular level, the modulation of expression profiles
of genes associated with the cellular apoptotic pathways upon activation was evaluated and the following three results have been demonstrated: (1)
activation results in selective resistance to apoptosis particularly to that induced by signaling via death
receptors and DNA damage; (2) concurrent with activation, the most apical protease in the death receptor
pathway, caspase-8/FLICE is rapidly down-regulated at the mRNA level representing a novel regulatory
mechanism; and (3) activation of monocytes also leads to dramatic induction of the Bfl-1 gene, an anti
apoptotic member of the Bcl-2 family. These findings thus provide a potential mechanistic basis for the
activation-induced resistance to apoptosis in human monocytes (Perera, 1998).
Sphingomyelinase (SMase) activation and ceramide generation have emerged as an important signaling pathway
transducing diverse biological effects of cytokine receptors like p55 tumor necrosis factor (TNF) receptor or
Fas. The TNF-dependent activation of acid SMase (A-SMase) through the p55 TNF
receptor-associated proteins TRADD and FADD is described. Overexpression of TRADD and FADD in 293 cells does not
change the basal activity of A-SMase but enhances TNF-induced stimulation of A-SMase. Other TNF
R55-associated proteins like TRAF2 and RIP, which have been reported to mediate TNF R55-mediated activation of
nuclear factor kappaB, do not affect activation of A-SMase. Caspase inhibitors markedly reduce A-SMase
activity, suggesting the involvement of an ICE-like protease in TRADD/FADD-mediated activation of A-SMase.
Overexpression of caspase-8/a (FLICE/MACH) or caspase-10/b (FLICE2) does not change A-SMase activity,
suggesting that TRADD/FADD-mediated activation of A-SMase involves a yet to be defined caspase-like
protease distinct from caspase-8/a or -10/b (Schwandner, 1998).
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