Death related ced-3/Nedd2-like protein
In comparison with other caspase family members, Drosophila caspase-1 is more homologous to CPP-32 and MCH-2alpha than to interleukin-1 beta-converting enzyme (ICE). It shares 37% sequence identity with both CPP-32 and MCH-2alpha, 29% identity with NEDD-2 (ICH-1), 28% homology with CED-2 and 25% homology with human ICE. This sequence similarity suggests that DCP-1 may be a member of the ced3-CPP-32 subfamily of caspases (Song, 1997).
A second Drosophila Caspase has been isolated and termed drICE. drICE is distinct from Death Caspase-1 and exhibits highest homology with the mammalian caspases, Mch2 and CPP32ß. drICE also contains a region in its putative small subunit that corresponds to the P4-specificity loop of CPP32ß. Overexpression of drICE sensitizes Drosophila cells to apoptotic stimuli; expression of an N-terminally truncated form of drICE rapidly induces apoptosis in Drosophila cells. Induction of apoptosis by reaper overexpression or by cycloheximide or etoposide treatment of Drosophila cells results in proteolytic processing of drICE. drICE is a cysteine protease that cleaves baculovirus p35 and Drosophila lamin DmO in vitro. drICE is expressed at all stages of Drosophila development at which programmed cell death can be induced. Levels are highest from 2-6 hours of embryogenesis, lower from 6-12 hours, and still lower after 12 hours of development. These results strongly argue that drICE is an apoptotic caspase that acts downstream of reaper (Fraser, 1997a).
The role of drICE was examined in in vitro apoptosis of the D. melanogaster cell line S2. Cytoplasmic lysates, made from S2 cells undergoing apoptosis induced by either reaper expression or cycloheximide treatment, contain a caspase activity with DEVD specificity that can cleave p35, lamin DmO, drICE and DCP-1 in vitro. This caspase activity can trigger chromatin condensation in isolated nuclei. Immunodepletion of drICE from lysates
is sufficient to remove most measurable in vitro apoptotic activity; re-addition of exogenous
drICE to such immunodepleted lysates restores apoptotic activity. It is concluded that, at least in S2
cells, drICE can be the sole caspase effector of apoptosis (Fraser 1997b).
Human CLARP, a caspase-like apoptosis-regulatory protein, contains two amino-terminal death effector domains fused to a carboxyl-terminal caspase-like domain. The structure and amino acid sequence of CLARP resembles those of caspase-8, caspase-10, and DCP2 (Caspase 2), a Drosophila melanogaster protein identified in this study. However, unlike caspase-8, two important residues predicted to be involved in catalysis are lost in the caspase-like domain of CLARP. Analysis with fluorogenic substrates for caspase activity confirms that CLARP is catalytically inactive. CLARP interacts with caspase-8 but not with FADD/MORT-1, an upstream death effector domain-containing protein of the Fas and tumor necrosis factor receptor 1 signaling pathways. Expression of CLARP induces apoptosis, which is blocked by the viral caspase inhibitor p35, dominant negative mutant caspase-8, and the synthetic caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-(OMe)-fluoromethylketone (zVAD-fmk). Moreover, CLARP augments the killing ability of caspase-8 and FADD/MORT-1 in mammalian cells. The human clarp gene maps to 2q33. Thus, CLARP represents a regulator of the upstream caspase-8, which may play a role in apoptosis during tissue development and homeostasis (Inohara, 1997).
To identify CAP3 and CAP4, components of the CD95 (Fas/APO-1) death-inducing signaling complex, nano-electrospray tandem mass spectrometry was used. This is a recently developed technique to sequence femtomole
quantities of polyacrylamide gel-separated proteins. Interestingly, CAP4 encodes a novel 55 kDa protein,
designated FLICE, which has homology to both FADD and the ICE/CED-3 family of cysteine proteases. FLICE
binds to the death effector domain of FADD and upon overexpression induces apoptosis that is blocked by the ICE
family inhibitors: CrmA and z-VAD-fmk. CAP3 was identified as the FLICE prodomain, which likely remains
bound to the receptor after proteolytic activation. Taken together, these data provide unique biochemical evidence to link a death
receptor physically to the proapoptotic proteases of the ICE/CED-3 family (Muzio, 1996).
Emerging evidence suggests that an amplifiable protease cascade consisting of multiple aspartate specific
cysteine proteases (ASCPs) is responsible for the apoptotic changes observed in mammalian cells undergoing
programmed cell death. Two novel ASCPs have been cloned from human Jurkat T-lymphocytes.
Like other ASCPs, the new proteases, named Mch4 and Mch5, are derived from single chain proenzymes.
However, their putative active sites contain a QACQG pentapeptide instead of the QACRG present in all known
ASCPs. Also, their N termini contain FADD-like death effector domains, suggesting possible interaction with
FADD. Expression of Mch4 in Escherichia coli produces an active protease that, like other ASCPs, is
potently inhibited by the tetrapeptide aldehyde DEVD-CHO. Interestingly, h Mch4 and the
serine protease granzyme B both cleave recombinant proCPP32 and proMch3 at a conserved IXXD-S sequence to
produce the large and small subunits of the active proteases. Granzyme B also cleaves proMch4 at a
homologous IXXD-A processing sequence to produce mature Mch4. These observations suggest that CPP32
and Mch3 are targets of mature Mch4 protease in apoptotic cells. The presence of the FADD-like domains in
Mch4 and Mch5 suggest a role for these proteases in the Fas-apoptotic pathway. In addition, these proteases
could participate in the granzyme B apoptotic pathways (Fernandes-Alnemri, 1996).
Fas (APO-1/CD95) is a transmembrane receptor protein that induces apoptosis upon activation. In apoptosis
triggered by Fas, a subset of cysteine proteases (designated caspases) is activated, playing a central role as
effector molecules. Among these caspases, human caspase-8 (FLICE/MACH/Mch5) has been isolated and
shown to be indispensable for Fas-mediated apoptotic signaling. The mouse
homolog to human caspase-8 has been isolated from a BaF3 cell cDNA library. This molecule conserves the death effector
domain (DED) and protease domain as detected in human caspase-8, and is capable of inducing apoptosis in
KB and Rat-1 cells when overexpressed. Expression of caspase-8 is detected by Northern-blot analysis in the various tissues of adult
mouse and in embryos at 9.5 days and 17.5 days of development. Further, a chromosomal gene for caspase-8 has been isolated from a mouse genomic library: the gene consists of eight exons and seven introns spanning about 26 kb in the coding region (Sakamaki, 1998).
A pivotal discovery established that Fas-associated death domain protein (FADD) interleukin-1beta-converting enzyme (FLICE)/MACH has a role in initiating the death pathway: this protease is recruited to the CD95 signaling complex by virtue of CD95's ability to bind the adapter molecule FADD. A new member of the caspase family has been cloned, a homolog of FLICE/MACH (Caspase 8), and Mch4. Since the overall architecture and function of this molecule is similar to that of FLICE, it has been designated FLICE2. Importantly, the carboxyl-terminal half of the small catalytic subunit that includes amino acids predicted to be involved in substrate binding is distinct. The pro-domain of FLICE2 encodes a functional death effector domain that binds to the corresponding domain in the adapter molecule FADD. Consistent with this finding, FLICE2 is recruited to both the CD95 and p55 tumor necrosis factor receptor signaling complexes in a FADD-dependent manner. A functional role for FLICE2 is suggested by the finding that an active site mutant of FLICE2 inhibits CD95 and tumor necrosis factor receptor-mediated apoptosis. FLICE2 is therefore involved in CD95 and p55 signal transduction (Vincenz, 1997).
Induction of apoptosis by the cell surface receptor CD95 (APO-1/Fas) has been shown to involve activation of a
family of cysteine proteases (caspases). The caspase FLICE is part of the CD95 death-inducing signaling complex and is therefore
the most upstream caspase in the CD95 apoptotic pathway. A total of eight different isoforms of FLICE
(caspase-8/a-h) have been described. To determine which isoforms are expressed in different cells a panel of monoclonal antibodies was generated, directed against all functional domains of FLICE. Using these
antibodies it has been shown that only two of the FLICE isoforms (caspase-8/a and caspase-8/b) are predominantly
expressed in cells of different origin. Both isoforms are recruited to the CD95 death-inducing signaling complex
and are activated upon CD95 stimulation with similar kinetics. Taken together, only two of the eight published
caspase-8 isoforms could be detected in significant amounts at the protein level (Scaffidi, 1997).
The death proteases caspase 8 (FLICE) and caspase 10 (Mch4/FLICE2) are recruited to
the CD-95 and tumor necrosis factor receptor-1 signaling complexes. This pivotal discovery suggested a mechanism used by these
cytotoxic receptors to initiate apoptosis. The cloning and characterization of I-FLICE is described, a
novel inhibitor of tumor necrosis factor receptor-1- and CD-95-induced apoptosis. The overall architecture of
I-FLICE is strikingly similar to that of FLICE and Mch4/FLICE2. However, I-FLICE lacks both a catalytic active
site and residues that form the substrate binding pocket, in keeping with its dominant negative inhibitory function.
I-FLICE is the first example of a catalytically inert caspase that can inhibit apoptosis (Hu, 1997).
Engagement of CD95 or tumor necrosis factor 1 receptor (TNFR-1) by ligand or agonist antibodies is capable of
activating the cell death program, the effector arm of which is composed of mammalian interleukin-1beta
converting enzyme (ICE)-like cysteine proteases (designated caspases) that are related to the Caenorhabditis
elegans death gene, CED-3. Caspases, unlike other mammalian cysteine proteases, cleave their substrates
following aspartate residues. Furthermore, proteases belonging to this family exist as zymogens, which in turn require
cleavage at internal aspartate residues to generate the two-subunit active enzyme. As such, family members are
capable of activating each other. Remarkably, both CD95 and TNFR-1 death receptors initiate apoptosis by
recruiting a novel ICE/CED-3 family member, designated FLICE/MACH, to the receptor signaling complex.
Therefore, FLICE/MACH represents the apical triggering protease in the cascade. Consistent with this,
recombinant FLICE is capable of proteolytically activating downstream caspases. Furthermore, CrmA, a
pox virus-encoded serpin that inhibits Fas and tumor necrosis factor-induced cell death attenuates the ability of
FLICE to activate downstream caspases (Muzio, 1997).
Viruses have evolved many distinct strategies to avoid the host's apoptotic response. A new
family of viral inhibitors (v-FLIPs) is described that interferes with apoptosis signaled through death receptors and which are
present in several gamma-herpesviruses (including Kaposi's-sarcoma-associated human herpesvirus-8), as well as
in the tumorigenic human molluscipoxvirus. v-FLIPs contain two death-effector domains that interact with the
adaptor protein FADD: this inhibits the recruitment and activation of the protease FLICE by the CD95 death
receptor. Cells expressing v-FLIPs are protected against apoptosis induced by CD95 or by the related death
receptors TRAMP and TRAIL-R. The herpesvirus saimiri FLIP is detected late during the lytic viral replication
cycle, at a time when host cells are partially protected from CD95-ligand-mediated apoptosis. Protection of
virus-infected cells against death-receptor-induced apoptosis may lead to higher virus production and contribute to
the persistence and oncogenicity of several FLIP-encoding viruses (Thome, 1997).
The binding of Fas ligand to Fas recruits caspase 8 to Fas via an adaptor, FADD/MORT1, and
activates a caspase cascade leading to apoptosis. A human Jurkat-derived cell line
(JB-6) is described that is deficient in caspase 8. This cell line is resistant to the apoptosis triggered by Fas
engagement. However, the multimerization of Fas-associated protein with death domain, through the
use of a dimerizing system, kills the JB-6 cells. This killing process is not accompanied by the
activation of caspases or DNA fragmentation. The dying cells show neither condensation nor
fragmentation of cells and nuclei, but the cells and nuclei swell in a manner similar to that seen in
necrosis. These results suggest that Fas-associated protein with death domain can kill the cells via
two pathways, one mediated by caspases and another that does not involve them (Kawahara, 1998).
Fas is a cell-surface receptor molecule that relays apoptotic (cell death) signals into cells. When Fas is activated by the binding of its ligand, the proteolytic protein
caspase-8 is recruited to a signaling complex known as DISC by binding to a Fas-associated adapter protein. A large new protein, FLASH, has now been
identified as a result of the cloning of its complementary DNA. This protein contains a motif with oligomerizing activity whose sequence is similar to that of the Caenorhabditis
elegans protein CED-4, and another domain (DRD domain) that interacts with a death-effector domain in caspase-8 or in the adapter protein. Stimulated Fas binds
FLASH, so FLASH is probably a component of the DISC signaling complex. Transient expression of FLASH activates caspase-8, whereas overexpression of a
truncated form of FLASH containing only one of its DRD or CED-4-like domains does not allow activation of caspase-8 and Fas-mediated apoptosis to occur.
Overexpression of full-length FLASH blocks the anti-apoptotic effect of the adenovirus protein E1B19K. FLASH is therefore necessary for the activation of
caspase-8 in Fas-mediated apoptosis (Imai, 1999).
The assembly of the CD-95 (Fas/Apo-1) receptor death-inducing signaling complex occurs in a hierarchical
manner; the death domain of CD-95 binds to the corresponding domain in the adapter molecule Fas-associated
death domain (FADD) Mort-1, which in turn recruits the zymogen form of the death protease caspase-8
(FLICE/Mach-1) by a homophilic interaction involving the death effector domains. Immediately after recruitment,
the single polypeptide FLICE zymogen is proteolytically processed to the active dimeric species composed of large
and small catalytic subunits. Since all caspases cleave their substrates after Asp residues and are themselves
processed from the single-chain zymogen to the two-chain active enzyme by cleavage at internal Asp residues, it
follows that an upstream caspase can process a downstream zymogen. However, since FLICE represents the most
apical caspase in the Fas pathway, its mode of activation has been enigmatic. It is hypothesized that the FLICE
zymogen possesses intrinsic enzymatic activity such that when approximated, it autoprocesses to the active
protease. Support for this is provided by two sources of evidence: (1) the synthesis of chimeric Fpk3FLICE molecules that can be
oligomerized in vivo by the synthetic cell-permeable dimerizer FK1012H2, and (2) cells transfected with Fpk3FLICE
undergo apoptosis after exposure to FK1012H2, producing a nonprocessable zymogen form of FLICE
that retains low but detectable protease activity (Muzio, 1998).
Many forms of apoptosis, including that caused by the death receptor CD95/Fas/APO-1, depend on the activation
of caspases, which are proteases that cleave specific intracellular proteins to cause orderly cellular disintegration.
The requirements for activating these crucial enzymatic mediators of death are not well understood. Using
molecular chimeras with either CD8 or Tac, it was found that oligomerization at the cell membrane powerfully induces
caspase-8 autoactivation and apoptosis. Death induction is abrogated by the z-VAD-fmk, z-IETD-fmk, or p35
enzyme inhibitors or by a mutation in the active site cysteine but is surprisingly unaffected by death inhibitor
Bcl-2 (Drosophila homolog: death executioner Bcl-2 homologue). Amino acid substitutions that prevent the proteolytic separation of the caspase from its membrane-associated
domain completely block apoptosis. Thus, oligomerization at the membrane is sufficient for caspase-8
autoactivation, but apoptosis could involve a death signal conveyed by the proteolytic release of the enzyme into the
cytoplasm (Martin, 1998).
Cytotoxic T lymphocytes induce apoptosis in target cells through the CD95(APO-1/Fas) and the perforin/granzyme
B (GrB) pathway. The exact substrate of GrB in vivo is still unknown, but to induce apoptosis GrB requires the
activity of caspases in target cells. In HeLa target cells induction of apoptosis through the
perforin/GrB pathway results in minor direct cleavage of CPP32 (caspase-3) by GrB. Most caspase-3 cleavage
results from activation of an upstream caspase. Moreover, target cells derived from caspase-3(-/-) mice display
GrB-induced poly(ADP-ribose) polymerase (PARP) cleavage with only partially reduced efficiency, as compared to
wild-type target cells. This indicates that other PARP-cleaving caspases can be activated during
perforin/GrB-induced cell death. In contrast to caspase-3, FLICE (caspase-8) is directly cleaved by GrB in HeLa
cells. It is therefore concluded that FLICE not only plays a central role in CD95(APO-1/Fas)-induced apoptosis but
can also be directly activated during perforin/GrB-induced apoptosis (Medema,1998).
The exit of cytochrome c from mitochondria into the cytosol has been implicated as an important step in
apoptosis. In the cytosol, cytochrome c binds to the CED-4 homolog, Apaf-1 (Drosophila homolog: Apaf-1-related-killer), thereby triggering
Apaf-1-mediated activation of caspase-9. Caspase-9 is thought to propagate the death signal by
triggering other caspase activation events, the details of which remain obscure. Six additional caspases (caspases-2, -3, -6, -7, -8, and -10) are processed in cell-free extracts in
response to cytochrome c, and three others (caspases-1, -4, and -5) fail to be activated under
the same conditions. In vitro association assays confirm that caspase-9 selectively binds to Apaf-1,
whereas caspases-1, -2, -3, -6, -7, -8, and -10 do not. Depletion of caspase-9 from cell extracts
abrogates cytochrome c-inducible activation of caspases-2, -3, -6, -7, -8, and -10, suggesting that
caspase-9 is required for all of these downstream caspase activation events. Immunodepletion of
caspases-3, -6, and -7 from cell extracts enables an ordering of the sequence of caspase activation events
downstream of caspase-9 and reveals the presence of a branched caspase cascade. Caspase-3 is
required for the activation of four other caspases (-2, -6, -8, and -10) in this pathway and also
participates in a feedback amplification loop involving caspase-9 (Slee, 1999).
Cytotoxic T lymphocytes induce apoptosis in target cells through the CD95(APO-1/Fas) and the
perforin/granzyme B (GrB) pathway. The exact substrate of GrB in vivo is still unknown, but to induce
apoptosis GrB requires the activity of caspases in target cells. In HeLa target cells
induction of apoptosis through the perforin/GrB pathway results in minor direct cleavage of CPP32
(caspase-3) by GrB. Most caspase-3 cleavage results from activation of an upstream caspase.
Moreover, target cells derived from caspase-3(-/-) mice display GrB-induced poly(ADP-ribose)
polymerase (PARP) cleavage with only partially reduced efficiency, when compared to wild-type target cells.
This indicates that other PARP-cleaving caspases can be activated during perforin/GrB-induced cell
death. In contrast to caspase-3, FLICE (caspase-8) is directly cleaved by GrB in HeLa cells. It is
therefore concluded that FLICE not only plays a central role in CD95(APO-1/Fas)-induced apoptosis
but that it can also be directly activated during perforin/GrB-induced apoptosis (Medema, 1997).
A phosphoprotein protein with a death effector domain has been characterized that has a novel bifunctional role in programmed cell death. The 15-kDa
phosphoprotein enriched in astrocytes (PEA-15) inhibits Fas-mediated apoptosis and increases tumor necrosis factor receptor-1 (TNF-R1)-mediated apoptosis in
the same cell type in a ligand-dependent manner. Phosphorylation appears to play a role in its differential effects, since point mutations at one or both
phosphorylation consensus sites within PEA-15 destroy its effect on Fas-mediated, but not TNF-R1-mediated, apoptosis. Furthermore, the differential effect is
evident at the level of caspase-8 activity, which is inhibited via Fas activation, but increased via TNF-R1 activation upon PEA-15 expression. These results show that
PEA-15 provides a potential mechanism during development for distinguishing between diverse extracellular death-inducing signals that culminate either in apoptosis
or in survival (Estelles, 1999).
PEA-15 mRNA is abundant as early as embryonic day 12. Changes in the phosphorylation state of PEA-15 in response to hormones and neurotransmitters
suggest that phosphorylation may be important to
the function of this protein. Although PEA-15 has a death effector domain (DED)
domain, the physiological role of PEA-15 and the significance of its phosphorylation state remain unclear. PEA-15 is unlike other DED-containing proteins described thus far in that its expression provides a means by which a
cell can respond differently to two extracellular apoptotic
stimuli. Such proteins are likely to serve an important role
during embryonic and adult development (Estelles, 1999).
Upon activation by liver injury, hepatic stellate cells produce excessive fibrous tissue leading to cirrhosis. The hepatotoxin CCl4 induces activation of RSK (see Drosophila RSK), phosphorylation of C/EBPß on Thr217, and proliferation of stellate cells in normal mice, but causes apoptosis of these cells in C/EBPß-/- or C/EBPß-Ala217 (a dominant-negative nonphosphorylatable mutant) transgenic mice. Both C/EBPß-PThr217 and the phosphorylation mimic C/EBPß-Glu217, but not C/EBPß-Ala217, associate with procaspases 1 and 8 in vivo and in vitro and inhibit their activation. These data suggest that C/EBPß phosphorylation on Thr217 creates a functional XEXD caspase substrate/inhibitor box (K-Phospho-T217VD) that is mimicked by C/EBPß-Glu217 (KE217VD). C/EBPß-/- and C/EBPß-Ala217 stellate cells are rescued from apoptosis by the cell permeant KE217VD tetrapeptide or C/EBPß-Glu217. It is concluded that C/EBPß phosphorylation by RSK creates a functional XEXD caspase inhibitory box critical for cell survival (Buck, 2001).
continued: Dredd Evolutionary homologs part 2/2 |
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