Death caspase-1
Targets of caspases (part 2/2) Certain cell types undergo apoptosis when they lose integrin-mediated contacts with
the extracellular matrix ("anoikis"). The Jun N-terminal kinase (JNK) pathway is
activated in and promotes anoikis. This activation requires caspase activity. A DEVD motif-specific caspase that specifically cleaves MEKK-1
is activated when cells lose matrix contact. The full length MEKK-1 is found in both attached and suspended cultured cells as a series of bands ranging from 160 kDa to 200 kDa, a hyperphosphorylated form. The C-terminal 80 kDa cleavage product increases significantly after suspension of the cells. Cleavage is required in order to activate the kinase.
When overexpressed, the MEKK-1 cleavage
product stimulates apoptosis; the wild-type, full-length MEKK-1 sensitizes cells to
anoikis; a cleavage-resistant mutant of MEKK-1 partially protects cells against
anoikis. The cleavage-resistant or kinase-inactive mutants also prevent caspase-7
from being activated completely. Thus, caspases can induce apoptosis by activating
MEKK-1, which in turn activates more caspase activity, comprising a positive
feedback loop (Cardone, 1997).
Apoptotic cells undergo characteristic morphological changes that include detachment of cell
attachment from the substratum and loss of cell-cell interactions. Attachment of cells to the
extracellular matrix and to other cells is mediated by integrins. The interactions of integrins with the
extracellular matrix activate focal adhesion kinase (FAK) and suppress apoptosis in diverse cell
types. Members of the tumor necrosis family such as Fas and Apo-2L, also known as tumor necrosis
factor-related apoptosis-inducing ligand (TRAIL), induce apoptosis in both suspension and adherent
cells through the activation of caspases. These caspases, when activated, cleave substrates that are
important for the maintenance of nuclear and membrane integrity. FAK is
sequentially cleaved into two different fragments early in Apo-2L-induced apoptosis. FAK cleavage is mediated by caspases, and FAK shows unique sensitivity to
different caspases. These results suggest that disruption of FAK may contribute to the morphological
changes observed in apoptotic suspension and adherent cells (Wen, 1997).
Members of the caspase-3 (CPP32, apopain, YAMA) family of cysteinyl proteases have been
implicated as key mediators of apoptosis in mammalian cells. Gelsolin has been identified as a substrate for caspase-3 by screening the translation products of small
complementary DNA pools for sensitivity to cleavage by caspase-3. Gelsolin is
cleaved in vivo in a caspase-dependent manner in cells stimulated by Fas.
Caspase-cleaved gelsolin severs the actin filaments in vitro in a Ca2+-independent
manner. Expression of the gelsolin cleavage product in multiple cell types causes the
cells to round up, detach from the plate, and undergo nuclear fragmentation.
Neutrophils isolated from mice lacking gelsolin have delayed onset of both blebbing and
DNA fragmentation, following apoptosis induction, as compared with wild-type
neutrophils. Thus, cleaved gelsolin may be one physiological effector of morphologic
change during apoptosis (Kothakota, 1997).
Proteolytic activation of hPAK65, a
p21-activated kinase, induces morphological changes and elicits apoptosis. hPAK65 is cleaved both in
vitro and in vivo by caspases at a single site between the N-terminal regulatory p21-binding domain and
the C-terminal kinase domain. The C-terminal cleavage product becomes activated, and exhibits a kinetic profile
that parallels caspase activation during apoptosis. This C-terminal hPAK65 fragment also activates the
c-Jun N-terminal kinase pathway in vivo. Microinjection or transfection of this truncated hPAK65 causes
striking alterations in cellular and nuclear morphology, which subsequently promotes apoptosis in both
CHO and Hela cells. Conversely, apoptosis is delayed in cells expressing a dominant-negative form of
hPAK65. These findings provide direct evidence that the activated form of hPAK65 generated by
caspase cleavage is a proapoptotic effector that mediates morphological and biochemical changes seen in
apoptosis (Lee, 1997).
Presenilin-2, the chromosome 1 familial Alzheimer's disease gene, has been shown to be involved in
programmed cell death by three complementary experimental approaches. Reduction of PS2 protein
levels by antisense RNA protects from apoptosis, whereas overexpression of an Alzheimer's PS2
mutant increases cell death induced by several stimuli. In addition, ALG-3, a truncated PS2 cDNA,
encodes an artificial COOH-terminal PS2 segment that dominantly inhibits apoptosis. A physiological COOH-terminal PS2 polypeptide (PS2s, Met298-Ile448) is described that is generated by both an
alternative PS2 transcript and proteolytic cleavage. PS2s protect transfected cells from
Fas- and tumor necrosis factor alpha (TNFalpha)-induced apoptosis. A similar
anti-apoptotic COOH-terminal PS2 polypeptide (PS2Ccas) is generated by caspase-3 cleavage at
Asp329. These results suggest that caspase-3 not only activates pro-apoptotic substrates but also
generates a negative feedback signal in which PS2Ccas antagonizes the progression of cell death.
Thus, whereas PS2 is required for apoptosis, PS2s and PS2Ccas oppose this process. The balance
between PS2 and these COOH-terminal fragments may dictate the cell fate (Vito, 1997).
Vitamin A and its derivatives, the retinoids, are essential regulators of many important biological
functions, including cell growth and differentiation, development, homeostasis, and carcinogenesis.
Natural retinoids such as all-trans retinoic acid can induce cell differentiation and inhibit growth of
certain cancer cells. A novel class of synthetic retinoids has been identified with strong anti-cancer
cell activities in vitro and in vivo which can induce apoptosis in several cancer cell lines. Using an
electrophoretic mobility shift assay, the DNA binding activity of several transcription
factors was examined in T cells treated with apoptotic retinoids. The DNA binding activity of the
general transcription factor Sp1 (see Drosophila Buttonhead) is lost in retinoid-treated T cells undergoing apoptosis. A truncated Sp1
protein is detected by immunoblot analysis, and cytosolic protein extracts prepared from apoptotic cells
contain a protease activity that specifically cleaves purified Sp1 in vitro. This proteolysis of Sp1 can
be inhibited by N-ethylmaleimide and iodoacetamide, indicating that a cysteine protease mediates
cleavage of Sp1. Inhibition of Sp1 cleavage by ZVAD-fmk and ZDEVD-fmk suggests
that caspases are directly involved in this event. In fact, caspases 2 and 3 are activated in T cells after
treatment with apoptotic retinoids. The peptide inhibitors also block retinoid-induced apoptosis, as
well as processing of caspases and proteolysis of Sp1 and poly(ADP-ribose) polymerase in intact cells.
Degradation of Sp1 occurs early during apoptosis and is therefore likely to have profound effects on
the basal transcription status of the cell. Interestingly, retinoid-induced apoptosis does not require de
novo mRNA and protein synthesis, suggesting that a novel mechanism of retinoid signaling is involved,
triggering cell death in a transcriptional activation-independent, caspase-dependent manner (Piedrafita, 1997).
A caspase-activated deoxyribonuclease (CAD) and its inhibitor (ICAD) have now been identified in the cytoplasmic fraction of mouse lymphoma cells. CAD is a protein of 343 amino acids that carries a nuclear-localization signal; ICAD exists in a long and a short form. Recombinant ICAD specifically inhibits CAD-induced degradation of nuclear DNA and its DNase activity. When CAD is expressed with ICAD in COS cells or in a cell-free system, CAD is produced as a complex with ICAD: treatment with caspase 3 releases the DNase activity that causes DNA fragmentation in nuclei. ICAD therefore seems to function as a chaperone for CAD during its synthesis, remaining complexed with CAD to inhibit its DNase activity; caspases activated by apoptotic stimuli then cleave ICAD, allowing CAD to enter the nucleus and degrade chromosomal DNA (Enari, 1998).
Caspase-activated DNase (CAD) cleaves chromosomal DNA during apoptosis. Two classes of human CAD cDNAs have been isolated from a human KT-3 leukemic cell cDNA library. One class of cDNA encodes a protein comprising 338 amino acids that shows a marked similarity to its murine counterpart. In vitro transcription and translation of this cDNA results in a functional CAD protein when the protein is synthesized in the presence of its inhibitor (inhibitor of CAD). The other cDNA class contains many deletions, insertions, and point mutations in the sequence corresponding to the coding region, suggesting that it is derived from a pseudogene. The functional CAD gene was localized to human chromosome 1p36.3 by fluorescent in situ hybridization. The CAD mRNA is expressed in a limited number of human tissues, including pancreas, spleen, prostate, and ovary. The expression of the CAD mRNA in human cell lines correlates with ability of the cells to show DNA fragmentation during apoptosis. Overexpression of CAD potentiates DNA fragmentation by apoptotic stimuli in these cell lines, indicating that CAD is responsible for the apoptotic DNA degradation (Mukae, 1998)
The transcription factor NF-kappaB is essential for survival of many cell types. However, cells can undergo apoptosis despite the concurrent NF-kappaB activation. It is unknown how the protection conveyed by NF-kappaB is overridden during apoptosis. IkappaB kinase (IKK) ß is specifically proteolyzed by Caspase-3-related caspases at aspartic acid residues 78, 242, 373, and 546 during tumor necrosis factor (TNF)-alpha-induced apoptosis. Proteolysis of IKKß eliminates its enzymatic activity, interfers with IKK activation, and promotes TNF-alpha killing. Point mutations that abrogate IKKß proteolysis generate a caspase-resistant IKKß mutant, which suppresses TNF-alpha-induced apoptosis. Thus, this study demonstrates that TNF-alpha-induced apoptosis requires caspase-mediated proteolysis of IKKß (Tang, 2001).
What is the mechanism by which the UC-IKKß mutant suppresses TNF-alpha-induced apoptosis? One of the possibilities is that it functions through prolonged induction of NF-kappaB-controlled antiapoptotic proteins. It is envisioned that apoptotic death of a cell requires amplification of the caspase cascade, an event that may depend on destruction of cellular survival factors. Treatment with TNF-alpha triggers rapid activation and reactivation of IKK and NF-kappaB. This results in production of antiapoptotic proteins, such as c-IAP1, which inhibit caspases. The life balance is maintained and cells survive. The addition of cycloheximide (CHX) reduces the production of antiapoptotic proteins, allowing activation of caspases such as Casp3. Activated Casp3 in turn cleaves IKKß, blunting IKK reactivation and subsequent production of antiapoptotic proteins. Activated Casp3 also cleaves antiapoptotic proteins, including c-IAP1. Therefore, the caspase cascade is amplified, switching the life balance toward death. In contrast, the uncleavable IKKß (IKK UCß) mutant is resistant to caspase-mediated proteolysis and can be reactivated over a prolonged period of time. Since 10 µg/ml CHX reduces, but does not completely block protein synthesis, activation of NF-kappaB by the UC IKKß mutant allows the accumulation of antiapoptotic proteins at a level that is sufficient to inhibit caspases. As a result, amplification of the caspase cascade and cell death is suppressed (Tang, 2001).
Caspases and the active phase of apoptosis Genetic studies have identified over a dozen genes that function in programmed cell death (apoptosis) in the nematode C. elegans. Although the
ultimate effects on cell survival or engulfment of mutations in each cell death gene have been extensively described, much less is known about how these mutations affect the kinetics of death and engulfment, or the interactions between these two processes. Four-dimensional-Nomarski time-lapse video microscopy has been used to follow in detail how cell death genes regulate the extent and kinetics of apoptotic cell death and removal in the early C. elegans embryo. Blocking engulfment enhances cell survival when cells are subjected to weak pro-apoptotic signals. Thus, genes that mediate corpse removal can also function to actively kill cells (Hoeppner, 2001).
In a weak caspase (ced-3) mutant background, cells at early stages (ring and erythrocyte stages) of cell death have three options: progression to full-blown apoptotic cell corpses, direct engulfment without morphological progression to full corpse, or reversion to normality and survival. It is proposed that the relative frequency of the latter two fates might be influenced by the efficiency with which the early corpse is recognized and engulfed. To determine whether preventing engulfment might enhance reversion from 'early death' and hence cell survival, the fate of the early AB cells was followed in ced-6;ced-3 and ced-7;ced-3 double mutant embryos. ced-6 and ced-7 are known to participate in the removal of apoptotic cells in C. elegans (Hoeppner, 2001).
Indeed, the absence of ced-6 or ced-7 function significantly increase the frequency of such reversion events. Furthermore, the fraction of cells that never initiated any overt signs of apoptosis is also greatly increased. How can the engulfment machinery contribute to cell killing? The data are consistent with at least two models. The 'backup-plan model' suggests that low levels of CED-3 caspase activity might on occasion be sufficient to activate the eat-me signal on the surface of the cell, but not enough to kill the cell. However, even if the cell does not autonomously kill itself, exposure of the eat-me signal ensures that it will be recognized and engulfed, and therefore properly removed. The alternative 'positive-feedback model' proposes that doomed cells indicate their desire to die to neighboring cells, either through cell-surface changes or secretion of a signaling molecule. Recognition of this signal results in the neighboring cells sending back pro-apoptotic signals, encouraging the doomed cell to 'go for it', thereby ensuring that
the cell completes the process. Completion of this positive feedback loop would somehow require an intact engulfment pathway, possibly for
transduction of the signal in the neighboring cells (Hoeppner, 2001).
Cytoplasmic extracts from chicken DU249 cells were compared at various stages along the apoptotic pathway. Extracts from
morphologically normal "committed stage" cells induce apoptotic morphology and DNA cleavage in substrate nuclei but require
ongoing caspase activity to do so. In contrast, extracts from frankly apoptotic cells induce apoptotic events in added nuclei in a
caspase-independent manner. Biochemical fractionation of these extracts reveals that a column fraction enriched in endogenous
active caspases is unable to induce DNA fragmentation or chromatin condensation in substrate nuclei, whereas a caspase-depleted
fraction induces both changes. Further characterization of the "execution phase" extracts reveals the presence of an ICAD/DFF45
(inhibitor of caspase-activated DNase/DNA fragmentation factor)- inhibitable nuclease resembling CAD, plus another activity that
is required for the apoptotic chromatin condensation. Despite the presence of active caspases, committed stage extracts lack
these downstream activities, suggesting that the caspases and downstream factors are segregated from one another in vivo during
the latent phase. These observations not only indicate that caspases act in an executive fashion, serving to activate downstream
factors that disassemble the nucleus rather than disassembling it themselves, but they also suggest that activation of the
downstream factors (rather than the caspases) is the critical event that occurs at the transition from the latent to active phase of
apoptosis (Samejima, 1998).
Caspases and development The developing cerebral cortex undergoes a period of substantial cell death. The present studies examine the role of the suicide receptor
Fas/Apo[apoptosis]-1 in cerebral cortical development. Fas mRNA and protein are transiently expressed in subsets of cells within the
developing rat cerebral cortex during the peak period of apoptosis. Fas-immunoreactive cells have been localized in close proximity to Fas ligand
(FasL)-expressing cells. The Fas-associated signaling protein receptor interacting protein (RIP) is expressed by some Fas-expressing cells,
whereas Fas-associated death domain (FADD) is undetectable in the early postnatal cerebral cortex. FLICE-inhibitory protein (FLIP), an
inhibitor of Fas activation, is also expressed in the postnatal cerebral cortex. Fas expression is more ubiquitous in embryonic cortical
neuroblasts in dissociated culture, as compared to in situ, within the developing brain, suggesting that the environmental milieu partly suppresses
Fas expression at this developmental stage. Furthermore, FADD, RIP, and FLIP are also expressed by subsets of dissociated cortical
neuroblasts in culture. Fas activation by ligand (FasL) or anti-Fas antibody induces caspase-dependent cell death in primary embryonic
cortical neuroblast cultures. The activation of Fas is also accompanied by a rapid downregulation of Fas receptor expression, non-cell
cycle-related incorporation of nucleic acids and nuclear translocation of the RelA/p65 subunit of the transcription factor NF-kappaB.
Together, these data suggest that adult cortical cell number may be established, in part, by an active process of receptor-mediated cell suicide,
initiated in situ by killer (FasL-expressing) cells and that Fas may have functions in addition to suicide in the developing brain (Cheema, 1999).
The epidermis is a multilayered squamous epithelium in which dividing basal cells withdraw from the
cell cycle and progressively differentiate as they are displaced toward the skin surface. Eventually, the
cells lose their nucleus and other organelles to become flattened squames, which are finally shed from
the surface as bags of cross-linked keratin filaments enclosed in a cornified envelope. Although
keratinocytes can undergo apoptosis when stimulated by a variety of agents, it is not known
whether their normal differentiation program uses any components of the apoptotic biochemical
machinery to produce the cornified cell. Differentiating keratinocytes have been reported to share
some features with apoptotic cells, such as DNA fragmentation, but these features have not been seen
consistently. Apoptosis involves an intracellular proteolytic cascade, mainly mediated by members
of the caspase family of cysteine proteases, which cleave one another and various key intracellular
target proteins to kill the cell neatly and quickly. Caspases are
activated during normal human keratinocyte differentiation and that activation is apparently
required for the normal loss of the nucleus. Intermediate-filament-associated protein filaggrin and its large precursor, profilaggrin normally accumulate in keratohyalin granules in the granular layer of the epidermis. Profilaggrin cleavage precedes nuclear degradation, and it plays an important part in the reorganization of the cytoskeleton that accompanies the formation of the cornified envelope. It is suggested that keratinocyte differentiation is accompanied by the activation of procaspase-3 and that the activation of at least one caspase is required for both nuclear loss and normal filaggrin processing (Weil, 1999).
The optical clarity of the lens is ensured by the programmed removal of nuclei and other organelles from the lens fiber cells during development. The morphology of
the degenerating nuclei is similar to that observed during apoptosis and is accompanied by DNA fragmentation. Proteins encoded by the bcl-2 proto-oncogene
family are important in either promoting or inhibiting apoptosis; caspases are involved in downstream proteolytic events. Here, the expression of bcl-2 family
members (bcl-2, bax, bad, and bcl-x[s/l]) and caspases-1, -2, -3, -4, and -6 was investigated through a range of stages of chick lens development using
immunocytochemistry, Western blotting, and affinity labeling for caspases using biotinylated caspase inhibitors. Using differentiating lens epithelial cell cultures, it has been
demonstrated that the addition to cultures of synthetic peptide inhibitors of caspases -1, -2, -4, -6, and -9 brings about a 50%-70% reduction in the number of
degenerating nuclei per unit area of culture, as assessed by image analysis. These effects were comparable to those seen when general inhibitors of caspases were
added to cultures. In contrast, the inhibitors of caspases-3 and -8 are not effective in significantly reducing the number of TUNEL-labeled nuclei. Expression of
the caspase substrates poly(ADP-ribose) polymerase (PARP) and the 45-kDa subunit of DNA fragmentation factor (DFF 45) was also observed in the developing
lens. Western blots of cultures to which caspase inhibitors are added reveal alterations in the PARP cleavage pattern, but not in that of DFF. These results
demonstrate a role for members of the bcl-2 family and caspases in the degeneration of lens fiber cell nuclei during chick secondary lens fiber development and
support the proposal that this process has many characteristics in common with apoptosis (Wride, 1999).
Activation of the protease caspase-3 is commonly thought to cause apoptotic cell death. Caspase-3 activity is regulated at postsynaptic sites in brain following stimuli associated with memory (neural activation and subsequent response habituation) instead of cell death. In the zebra finch auditory forebrain, the concentration of caspase-3 active sites increases briefly within minutes after exposure to tape-recorded birdsong. With confocal and immunoelectron microscopy, the activated enzyme was localized to dendritic spines. The activated caspase-3 protein is present even in unstimulated brain but bound to an endogenous inhibitor, BIRC4 (xIAP), suggesting a mechanism for rapid release and sequestering at specific synaptic sites. Caspase-3 activity is necessary to consolidate a persistent physiological trace of the song stimulus, as demonstrated using pharmacological interference and the zenk gene habituation assay. Thus, the brain appears to have adapted a core component of cell death machinery to serve a unique role in learning and memory (Huesmann, 2006; full text of article).
This study describes a series of studies designed to assess the role of caspase-3 activity in the phenomenon of song-specific habituation in adult zebra finches. This is an especially favorable model for analyzing biochemical changes associated with memory formation. A large discrete area in the forebrain mediates the representation of songs. When a bird hears the same song repeatedly in the same context, the neurophysiological response to that specific song habituates; this habituation can persist for days or even longer. Song presentation also triggers robust molecular responses in this area, which also change as the presentation is repeated. Novel songs initially activate the ERK intracellular signaling pathway, followed by a pulse of zenk gene transcription (Kruse, 2000; Mello, 1992).
zenk is an immediate early gene known as zif-268, egr-1, NGFI-A, or Krox-24, referred to by the acronym 'ZENK'. When the stimulus is repeated across an hour or more, these molecular responses themselves habituate without affecting the responses to other songs. Habituation of the zenk response to a song is correlated with emergence of a persistent change in the behavioral response to that song. The zenk gene response is especially easy to measure and thus zenk gene expression in the auditory forebrain may be used as a molecular indicator of the status of a particular contextual song memory. If the song is heard as 'novel' it induces a zenk response; after the song has been entrained it no longer induces zenk (Huesmann, 2006).
This study shows novel-song exposure also triggers a rapid and transient increase in immunoreactivity for the activated form of caspase-3 and that caspase-3 activity is necessary for development of long-term habituation. The increase is specifically localized to postsynaptic terminals within the auditory forebrain, and evidence is provided for a molecular mechanism that could account for this tight temporal and anatomical control. These results establish a key role for caspase-3 in the machinery of memory consolidation (Huesmann, 2006).
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