From the Department of Pathology, University of Washington, Seattle.
Correspondence to Stephen M. Schwartz, Department of Pathology, University of Washington, Box 357335, Seattle, WA 98195-7335. E-mail steves{at}u.washington.edu
The
article by Yaoita et al1 in this issue of
Circulation is the first of what will indubitably be many
articles on the role of the caspases in cell death. This is an
important event. Until 10 years ago, we could not define death at all.
Instead, we relied on the process of necrosis, the decay of the cell
after its death, as a way to tell us that cells had died. Typical
experiments involved applying a death stimulus for different time
periods and then waiting hours or days to see if the death stimulus had
effected a critical "point of no return."
This point of view changed dramatically starting with the work of
Horvitz in Caenorhabditis elegans. He defined three genes
that made up a genetic pathway determining cell death in the nematode.
These three genes were called ced-3, ced-4, and
ced-9. ced-3 turned out to be a cysteine
protease, that is, a protease with –SH in its active site instead of
serine's –OH. Today we know that there are at least 10 caspases, the
new name for cysteine proteases, and these form a cascade controlling
death in most situations studied thus far.2 3
The control of the caspases seems to depend on a simple principle: the
enzymes are normally inactive as proforms. Activation requires
proteolytic cleavage of the caspases at specific sites, and in most
cases, these sites are themselves substrates for caspases. So, by
analogy to coagulation, death is controlled by a cascade of proteases
acting on each other.
The promise of this as a therapeutic pathway emerges from two sources.
First, a number of investigators, mainly in companies, have developed
low-molecular-weight protease inhibitors. The specificity
of these, as shown in the Figure
Another reason for believing in the caspases as a likely therapeutic
target is the recent evidence that cells not only have caspases, they
also have proteins able to inhibit the caspases or prevent their
activation. The serine protease coagulation and complement cascades are
regulated by antiproteases, including proteases that digest and
inactivate other proteases, allosteric modifiers of
protease activity or irreversible protease substrates (serpins) that
bind and inactivate enzymes. Examples of similar
"anti-caspases" are now emerging for the caspases
(Table
The simplest category of inhibitors may be substrates
themselves. Tatsuta et al6 made the intriguing
observation that cytoplasmic interleukin-1ß (IL-1ß), the
prototypical substrate of the caspases, can act to inhibit Fas-mediated
cell death. Although this might be an artifact of IL-1ß secretion, it
is worth considering the possibility that low levels of turnover of
substrate regulate the activity of the low abundance, long-prodomain
caspases and account for the need for activation of downstream caspases
before death can occur.
Another category of caspase inhibitors depends on the
activation mechanism for the long-prodomain caspases. Caspases 8 and 10
are activated by interacting with a death adapter protein,
FADD. FADD aggregates these "signaling" caspases onto the Fas
receptor after the receptor aggregates as a result of interacting with
its ligand. For example, sentrin is a protein that binds domains on Fas
but not FADD. Sentrin may inhibit FADD-dependent death by preventing
aggregation of FADD on activated Fas and secondarily inhibiting
recruitment of caspase 8.7
Like the coagulation enzymes, the caspases also appear to be regulated
by serpins. Viral serpins that enhance viral survival by inhibiting
caspases include p35, a general inhibitor of caspases by
viruses in insect cells, and CrmA, produced by the cowpox virus in
mammalian cells. Recently, an endogenous, nonviral serpin
for the caspases has been identified in mammalian cells, proteinase
inhibitor 9 (PI9). Sprecher and
collaborators8 used serpin homology to clone PI9.
Other members of this new family of nonsecreted, cytoplasmic serpins
have been serine protease
inhibitors9 10 ; however PI9 is a CrmA
homolog. The selectivity of PI9 could be relevant to recent
observations that different caspases, especially the long prodomain
caspases including caspases 2, 8, 9, and 10, are involved in different
types of cell death. Because PI9 can inhibit interleukin-1–converting
enzyme (ICE), it is possible that it plays a role in inhibition of the
ICE-like caspase believed to be required to activate
mitochondria.11 Recently, Schonbeck et
al12 reported an as-yet-unidentified protease
inhibitor capable of blocking caspase 1 (ICE) in smooth
muscle but not endothelial cytoplasm.
The next category of caspase inhibitors, called IAPs, were
also first recognized as viral proteins.13 14 15 16 17 18 19
Recently, however, mammalian IAPs have been recognized as well. Binding
of IAPs to the TRAF molecules in the death receptor complexes suggested
a role at the level of the initial activation of the long-prodomain
caspases; however, a recent paper by Deveraux et
al20 found that a mammalian X-linked IAP, XIAP,
inhibited death by binding to caspases 3 and 7. Intriguingly, this
effect was relatively specific for these terminal caspases. XIAP did
not activate caspases 8, 6, or 1 even at 50-fold excess, at
least against the substrate tested. This suggests that XIAP is an
inhibitor of specific caspases. The mechanism of protease
inhibition is not apparent, and it is not known whether this effect is
limited to this one member of the IAP family.
The final category of caspase inhibitors identified first
in viruses are called "FLIPs." The FLIPs were recently discovered
by Thome et al21 and Hu et
al22 as viral proteins with homology to the DED
of caspase 8 (FLICE). Death effector domains are the domains caspases
use to aggregate to one another and to FADD. Like DED constructs of
caspase 8,21 the viral FLIPs act as dominant
negatives for FADD-mediated death apparently by acting as competitors
for binding of the prodomains of caspase 8 or 10 and thus blocking
Fas-mediated apoptosis. In recent months, eight different labs
including our own have cloned cellular homologs of the FLIP
gene.23 We called this cellular FLIP "MRIT."
Intriguingly, MRIT abundance is very high in
myocardium.
Until this point, I have focused on the anticaspases as
antiapoptotics. However, most readers are likely to be more
familiar with another category of antiapoptotic genes, the
"Bcl2" family. This is the same family as the
ced-9 gene already referred to in my comments about
Horvitz's work in C. elegans. Bcl2 members do
control death, and ced-9 appears to be mainly
antiapoptotic. The mechanism for this action is confusing
because Bcl2 homologs can also stimulate cell death. The
proapoptotic Bcl2 homologs are believed to possess
this activity via their ability to increase mitochondrial permeability,
releasing the cytochrome c. Bcl2 and its
proapoptotic relatives seem to determine mitochondrial
permeability by competing with each other in the formation of
mitochondrial pores or perhaps by interacting with other molecules,
including the mammalian ced-4, to control caspase activation
(see below). Cytochrome c in turn activates a
cytoplasmic molecule that we now know is the mammalian equivalent of
Horvitz's third gene, ced-4. ced-4 functions,
when complexed with cytochrome c, to activate the
effector caspases.24 25 26 27 28 29 30 31 32 33 34 In summary, we are
beginning to see a central pathway of death that centers on the caspase
cascade.
In this regard, the article by Yaoita et al1 is
likely to be the first of many exploring a new therapeutic direction. I
would suggest there are three key areas to consider:
1. Tissue specificity. The first generation of drugs are very
nonspecific, and we know little about their possible toxicity, even in
animals.
2. Therapeutic efficacy. It is important to remember that caspases are
not synonymous with death. Although death may be mediated in many
situations by the effector caspases, it is likely that not all death
occurs this way and equally likely that caspases play a role in
cellular events in addition to death itself. An intriguing example of
the latter may be the recent paper by Rodriguez et
al.35 Normally, infusion of Fas-activating
antibodies kills an animal because the liver is very sensitive to Fas
activation. However, when these investigators protected the liver with
an antiapoptotic transgene, the mice still died. The mice
showed no morphological indication of apoptosis, raising the
intriguing possibility that death may have occurred from a sublethal
activation of the caspase pathway (at least, sublethal at the level of
the individual cell).
3. Finally, as seen in the article by Yaoita et
al1 in this issue of Circulation, the
protective effect of ZVAD-fmk was not total. Moreover, even the
morphological evidence of an antiapoptotic effect, measured by
the terminal deoxynucleotidyl transferase–mediated
dUTP-biotin nick end labeling assay, correlated poorly and was much
more marked than the more functional measure of infarcted area. We do
not know whether this is an issue of dose or an issue of the duration
of injury versus the duration of drug.
Footnotes
The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
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© 1998 American Heart Association, Inc.
Editorials
Cell Death and the Caspase Cascade
Key Words: Editorials • apoptosis • cells • proteins
, is
still broad, but it is likely that more specific inhibitors
will emerge. Second, we already know that there is an order to the
pathway. For example, caspases 8 and 10 are "long-prodomain"
activating caspases. They exert their activity on death not directly,
but by interacting with specific receptors. These receptors include Fas
and such Fas-related proteins as the tumor necrosis factor-1
receptor. It is likely that specific long-prodomain caspases will be
found to interact with different death stimuli. Similarly, the caspases
include short-prodomain caspases, eg, 3, 6, and 7. These seem to be the
targets of the long-prodomain receptors and mediate the final
proteolytic steps in the death pathway.4 5
Intriguingly, even knockouts in the effector caspases have a limited
phenotype. This offers tremendous promise for our ability in
the future to develop drugs appropriate to different kinds of
death.
View larger version (61K):
[in a new window]
Figure 1. Classification of caspases. The shaded "U" encloses
caspases believed to be the final mediators of death. Death may be
initiated by receptors interacting with DED long-prodomain caspases and
by mitochondria. ).
View this table:
[in a new window]
Table 1. Anticaspases
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