caspases coordinate , in a proteolytic cascade and with the help of other proteases , the rapid and efficient elimination of a cell by apoptosis .
they are classified into monomeric initiator caspases ( caspase-8 and -9 ) with long prodomains ( death effector domain [ ded ] , and caspase recruitment domain [ card ] ) , which are activated by recruitment to and dimerization on protein platforms , and already dimerized effector caspases ( caspase-3 , -6 and -7 ) , which require for their activation the cleavage ( e.g. , by initiator caspases ) of an intersubunit linker that binds to the dimer interface . in the extrinsic signaling pathway ,
the recruitment platform is the death - inducing signaling complex ( disc ) consisting of the activated death receptor , the adapter fadd ( fas - associated protein with death domain ) and initiator caspase-8 and -10 . in the intrinsic mitochondrial pathway ,
the platform is the apoptosome , a complex of the adapter apaf-1 ( apoptotic protease activating factor 1 ) , mitochondrially - released cytochrome c and initiator caspase-9 . in both cases ,
the main role of caspase-8 and -9 is to cleave and activate effector caspase-3 and -7 .
caspase-6 is an effector caspase with a limited substrate specificity whose exact role in apoptosis remains ill - defined .
caspase-2 is recruited to another platform , the piddosome ( pidd is the p53-induced protein with death domain ) , which is involved in translating a danger signal , such as dna damage , into either a repair or death response depending on the cellular or environmental context .
similarly , caspases-1 , -4 and -5 in humans and caspase-11 in mice are part of inflammasome platforms that assemble in response to external , pathogenic stimuli and guide the production and secretion of pro - inflammatory cytokines such as interleukin ( il)-1 and il-18 . under certain circumstances , such as salmonella - infected macrophages ,
the antimicrobial , inflammatory response may result in a caspase-1-mediated form of programmed cell death , called pyroptosis .
caspase-12 is an inhibitor of caspase-1 , and caspase-14 is probably the only truly non - apoptotic human caspase mediating keratinocyte differentiation . in the caspase field ,
are non - caspase proteases important to support or even replace caspase - mediated signaling ? and
why is effector caspase activation and apoptosis not synchronized , even in a clonal cell population ?
two recent papers reported on the caspase substrate repertoire in apoptotic cells . in one case , the proteomes from control and apoptosis - stimulated systems were separated on one - dimensional sds - page ( sodium dodecyl sulfate polyacrylamide gel electrophoresis ) and analyzed by liquid chromatography coupled with tandem mass spectrometry ( lc - ms / ms ) after gel elution . in the other approach
a gel - free technology was used by selectively biotinylating free protein amino termini that are generated in apoptotic cells upon caspase - mediated cleavages to enrich for lc - ms / ms .
both groups identified hundreds of new substrates and support the concept that most of them are cleaved into domain - containing fragments that may either gain or lose function .
the cleaved substrates are often stable , suggesting that dismantling the apoptotic cell is more like folding a tent after careful removal of pegs than disposing debris after an explosion .
they found that caspase-3 was more promiscuous than caspase-7 because 12 out of 20 substrates were preferentially cleaved by caspase-3 whereas only one was more susceptible to processing by caspase-7 .
thus , caspase-3 and -7 may , in part , be functionally distinct , explaining why caspase-3 and caspase-7 null mice show distinct phenotypes on some genetic backgrounds . in this regard , a recent study published for the first time a non - redundant role of caspase-7 in mediating lipopolysaccharide - induced lymphocyte apoptosis and mortality in mice .
this might be due to the fact that caspase-7 , unlike caspase-3 , can be processed and activated by caspase-1 .
thus , under certain conditions caspase-7 may be crucial for inflammatory events triggered by the caspase-1 inflammasome .
caspase-3 can also process other caspases , such as caspase-6 and -2 . in turn ,
caspase-6 processes caspase-8 and -10 . using a specific inhibitor of caspase-3 , and small interfering rna - mediated ablation of specific caspases , inoue et al .   recently validated this caspase ordering pathway not only for caspase-3 but also for caspase-7 .
caspase-2 was previously suggested to function as an initiator caspase for dna damage and heat - shock - induced apoptosis upstream of mitochondria .
it seems that caspase-2 may amplify apoptotic responses by being activated downstream of caspase-9/-3   or , alternatively , to drive a dna damage response that bypasses p53 , b - cell lymphoma protein-2 ( bcl-2 ) and caspase-3 .
moreover , a non - apoptotic role of caspase-2 was proposed in the context of dna damage - associated cell cycle arrest through the recruitment and activation on a cytosolic and a nuclear piddosome platform   because caspase-2 deficient cells failed to show g2/m arrest , leading to more mitotic cells whose dna was not repaired . in accordance with such a mechanism , ho et al .  
since the number of therapies associated with increased or decreased apoptosis continue to grow , it is crucial to directly visualize and quantify apoptosis in vivo .
developed fluorescently - labeled activity - based probes ( abps ) against caspases that provided direct readouts of kinetics of apoptosis in thymi and tumors in live mice .
in contrast to the already - used polymer - based quenched fluorescent substrates that are cleaved in multiple locations to produce fluorescent products , the abps are retained at the site of proteolysis by forming a covalent bond with the active - site cysteine of caspases .
thus , the fluorescent abps show more rapid and selective uptake into tumors and brighter signals compared to substrate probes .
human diseases characterized by high apoptosis resistance , such as cancer , may be treated in the future with small - molecule drugs that can activate effector caspase proenzymes .
wolan et al .   used high - throughput screening to identify a compound that promoted selective autoproteolytic activation of pro - caspase-3 at physiological concentrations by stabilizing the on - state conformation of the proenzyme .
moreover , walters et al .   recently reported on a caspase-3 mutation that prevented the intersubunit linker from binding in the dimer interface , allowing the active site to form in the procaspase in the absence of proteolysis .
this pseudoactivated caspase-3 was not inhibited by xiap ( x - linked inhibitor of apoptosis protein ) and rapidly killed cells at low concentrations . in mammalian cells
this form of cell death seems to be mediated by other proteases , such as calpains , serine proteases or lysosomal proteases , which act in parallel with caspases to amplify apoptosis signaling or induce other forms of cell death such as necrosis .
calpains mainly play a pro - death role when calcium signaling is involved . in 2005 ,
nicotera s group showed that the plasma membrane na / ca exchanger is cleaved by calpain in the ischemic brain and in cerebellar granule neurons exposed to glutamate leading to a ca - mediated neuronal demise .
moreover , calpains were recently shown to redistribute to the nuclear envelope , where they degraded nuclear pore complexes and increased nuclear leakiness in response to a ca overload .
furthermore , calpain-1 was found to be activated in spontaneous and fas - mediated apoptosis of neutrophils by a caspase - induced cleavage of its inhibitor calpastatin .
this resulted in the truncation of pro - apoptotic bax ( bcl-2-associated x protein )   and the autophagy - related 5 ( atg5 ) protein , which both enhanced cytochrome c release and most likely deviated the cells from autophagy to apoptosis .
masson and tschopp   identified a family of serine proteases , the granzymes ( gzms ) , present in cytotoxic t cells and natural killer cells , which were capable of eliminating virally infected and malignant cells in cooperation with the fasl - fas signaling system .
meanwhile , it has become clear that gzmb mediates caspase - dependent apoptosis by either cleaving bid ( bh3-interacting domain death agonist ) and activating bax / bak - dependent cytochrome c release or directly processing caspase-3 .
by contrast , metkar et al .   recently reported that gzma contributes to anti - viral immunity , not via its pro - apoptotic action , but by triggering the processing of pro - il-1 to mature il-1 , thereby stimulating a pro - inflammatory response .
thus , gzms resemble caspases in their capacity to influence both apoptosis and inflammation depending on the isoform . in this respect ,
the orphan human gzmm and gzmk may similarly act as pro - inflammatory proteases , explaining the observed ongoing anti - viral defence in gzmaxb-/- mice .
moreover , it is conceivable that other cell types may also exploit a serine protease - based mechanism to regulate apoptosis .
a few candidate serine proteases have so far been proposed , such as htra2 ( high - temperature - regulated a2 ) , ap24 ( 24 kda apoptotic protease ) and serine proteases implicated in endoplasmatic reticulum stress - induced apoptosis .
cathepsins encompass three classes of lysosomal proteases , the serine proteases cathepsin a and cathepsin g , the aspartic proteases cathepsin d and cathepsin e and the 11 so far known human cysteine proteases , cathepsins b , c , f , h , k , l , o , s , v , w and x / z .
they have been implicated in apoptosis regulation because of their cytoplasmic release due to increased lysosomal membrane permeabilization ( lmp ) .
although they depend on acidic ph for optimal activity , they seem to retain some of their catalytic activity at physiological ph .
therefore , upon their release into the cytoplasm they can directly cleave caspases   or , as recently reported , pro- and anti - apoptotic members of the bcl-2 family or xiap . unfortunately , in most studies , lmp was artificially induced by lysosomotropic agents such as leu - leu - ome ( l - leucyl - l - leucine methyl ester ) , and it has therefore remained unclear if cathepsin release indeed plays an initiating role in apoptosis signaling .
report that cells deficient in bax / bak , apoptosomal components or caspase-3/-7 are resistant to lmp in response to various apoptotic stimuli , indicating that cathepsin release and action occurs downstream of mitochondria . moreover , since some cathepsin knock - out cells ( b , l but not d ) exhibited delayed but not blocked apoptosis , this lysosomal signaling pathway seems to amplify rather than initiate apoptosis .
this process could nevertheless be physiologically relevant because a lack of this amplifcation loop would lead to delayed cell death and/or inefficent removal of apoptotic bodies and may be the underlying cause of a lack of resolution of inflammation due to neutrophil accumulation in cathepsin d knock - outs , reduced keratinocyte apoptosis resulting in periodic hair loss in cathepsin l - deficient mice   and diminished liver damage upon tumor necrosis factor ( tnf)-alpha challenge in mice deficient of cathepsin b .
more recently , kirkegaard et al .   offered a possibility to stabilize lysosomes by delivering hsp70 ( heat - shock protein 70 ) via the endocytotic pathway . through its interaction with an endolysosomal phospholipid
this could be exploited to correct niemann - pick disease - associated pathology ( in which acid spingomyelinase is defective ) .
conversely , as tumor cells express high levels of lysosomal hsp70 , a reduction of this protein may sensitize these cells to lmp and cathepsin - mediated cell death .
it has remained enigmatic why cells , even in clonal populations , undergo apoptosis at different rates despite simultaneous addition of an apoptotic stimulus .
although caspase-3 activation occurs within minutes after cytochrome c release , the duration of events preceding cytochrome c release can vary from 1 to 24 hours .
a number of factors could contribute to this population variability , including differences in cell cycle stages , metabolic rates , the local environment of single cells or stochasticity in biochemical reactions triggered by apoptotic stimuli .
now , two recent papers reveal that the cell - cell variability of apoptosis times is due to the divergence of the molecular composition of the cells .
using a novel single fluorophore reporter to measure caspase-3/-7 activities inside cells , bhola et al .  
report that the onset of proteolytic activity is tightly synchronized amongst the most related sister cells and lost over successive generations .
similiarly , spencer et al .   found that variability in the apoptotic phenotype arises from cell - to - cell differences in protein levels of caspases , caspase inhibitors , bcl-2 family members , and so on , that exist before trail ( tnf - related apoptosis - inducing ligand ) exposure .
a particular protein state of a single cell is then transmitted to the daughter cells , giving rise to transient non - genetic heritability in cell fate .
due to ongoing protein synthesis , the protein content of sister cells rapidly diverges so that soon these cells are no more similar to each other than pairs of cells chosen at random .
two recent papers reported on the caspase substrate repertoire in apoptotic cells . in one case , the proteomes from control and apoptosis - stimulated systems were separated on one - dimensional sds - page ( sodium dodecyl sulfate polyacrylamide gel electrophoresis ) and analyzed by liquid chromatography coupled with tandem mass spectrometry ( lc - ms / ms ) after gel elution . in the other approach
a gel - free technology was used by selectively biotinylating free protein amino termini that are generated in apoptotic cells upon caspase - mediated cleavages to enrich for lc - ms / ms .
both groups identified hundreds of new substrates and support the concept that most of them are cleaved into domain - containing fragments that may either gain or lose function .
the cleaved substrates are often stable , suggesting that dismantling the apoptotic cell is more like folding a tent after careful removal of pegs than disposing debris after an explosion .
they found that caspase-3 was more promiscuous than caspase-7 because 12 out of 20 substrates were preferentially cleaved by caspase-3 whereas only one was more susceptible to processing by caspase-7 .
thus , caspase-3 and -7 may , in part , be functionally distinct , explaining why caspase-3 and caspase-7 null mice show distinct phenotypes on some genetic backgrounds . in this regard , a recent study published for the first time a non - redundant role of caspase-7 in mediating lipopolysaccharide - induced lymphocyte apoptosis and mortality in mice .
this might be due to the fact that caspase-7 , unlike caspase-3 , can be processed and activated by caspase-1 .
thus , under certain conditions caspase-7 may be crucial for inflammatory events triggered by the caspase-1 inflammasome .
caspase-3 can also process other caspases , such as caspase-6 and -2 . in turn ,
caspase-6 processes caspase-8 and -10 . using a specific inhibitor of caspase-3 , and small interfering rna - mediated ablation of specific caspases , inoue et al .   recently validated this caspase ordering pathway not only for caspase-3 but also for caspase-7 .
caspase-2 was previously suggested to function as an initiator caspase for dna damage and heat - shock - induced apoptosis upstream of mitochondria .
it seems that caspase-2 may amplify apoptotic responses by being activated downstream of caspase-9/-3   or , alternatively , to drive a dna damage response that bypasses p53 , b - cell lymphoma protein-2 ( bcl-2 ) and caspase-3 .
moreover , a non - apoptotic role of caspase-2 was proposed in the context of dna damage - associated cell cycle arrest through the recruitment and activation on a cytosolic and a nuclear piddosome platform   because caspase-2 deficient cells failed to show g2/m arrest , leading to more mitotic cells whose dna was not repaired . in accordance with such a mechanism , ho et al .  
since the number of therapies associated with increased or decreased apoptosis continue to grow , it is crucial to directly visualize and quantify apoptosis in vivo .
developed fluorescently - labeled activity - based probes ( abps ) against caspases that provided direct readouts of kinetics of apoptosis in thymi and tumors in live mice .
in contrast to the already - used polymer - based quenched fluorescent substrates that are cleaved in multiple locations to produce fluorescent products , the abps are retained at the site of proteolysis by forming a covalent bond with the active - site cysteine of caspases .
thus , the fluorescent abps show more rapid and selective uptake into tumors and brighter signals compared to substrate probes .
human diseases characterized by high apoptosis resistance , such as cancer , may be treated in the future with small - molecule drugs that can activate effector caspase proenzymes .
wolan et al .   used high - throughput screening to identify a compound that promoted selective autoproteolytic activation of pro - caspase-3 at physiological concentrations by stabilizing the on - state conformation of the proenzyme .
moreover , walters et al .   recently reported on a caspase-3 mutation that prevented the intersubunit linker from binding in the dimer interface , allowing the active site to form in the procaspase in the absence of proteolysis .
this pseudoactivated caspase-3 was not inhibited by xiap ( x - linked inhibitor of apoptosis protein ) and rapidly killed cells at low concentrations .
this form of cell death seems to be mediated by other proteases , such as calpains , serine proteases or lysosomal proteases , which act in parallel with caspases to amplify apoptosis signaling or induce other forms of cell death such as necrosis .
calpains mainly play a pro - death role when calcium signaling is involved . in 2005 ,
nicotera s group showed that the plasma membrane na / ca exchanger is cleaved by calpain in the ischemic brain and in cerebellar granule neurons exposed to glutamate leading to a ca - mediated neuronal demise .
moreover , calpains were recently shown to redistribute to the nuclear envelope , where they degraded nuclear pore complexes and increased nuclear leakiness in response to a ca overload .
furthermore , calpain-1 was found to be activated in spontaneous and fas - mediated apoptosis of neutrophils by a caspase - induced cleavage of its inhibitor calpastatin .
this resulted in the truncation of pro - apoptotic bax ( bcl-2-associated x protein )   and the autophagy - related 5 ( atg5 ) protein , which both enhanced cytochrome c release and most likely deviated the cells from autophagy to apoptosis .
masson and tschopp   identified a family of serine proteases , the granzymes ( gzms ) , present in cytotoxic t cells and natural killer cells , which were capable of eliminating virally infected and malignant cells in cooperation with the fasl - fas signaling system .
meanwhile , it has become clear that gzmb mediates caspase - dependent apoptosis by either cleaving bid ( bh3-interacting domain death agonist ) and activating bax / bak - dependent cytochrome c release or directly processing caspase-3 .
by contrast , metkar et al .   recently reported that gzma contributes to anti - viral immunity , not via its pro - apoptotic action , but by triggering the processing of pro - il-1 to mature il-1 , thereby stimulating a pro - inflammatory response .
thus , gzms resemble caspases in their capacity to influence both apoptosis and inflammation depending on the isoform .
in this respect , the orphan human gzmm and gzmk may similarly act as pro - inflammatory proteases , explaining the observed ongoing anti - viral defence in gzmaxb-/- mice .
moreover , it is conceivable that other cell types may also exploit a serine protease - based mechanism to regulate apoptosis .
a few candidate serine proteases have so far been proposed , such as htra2 ( high - temperature - regulated a2 ) , ap24 ( 24 kda apoptotic protease ) and serine proteases implicated in endoplasmatic reticulum stress - induced apoptosis .
cathepsins encompass three classes of lysosomal proteases , the serine proteases cathepsin a and cathepsin g , the aspartic proteases cathepsin d and cathepsin e and the 11 so far known human cysteine proteases , cathepsins b , c , f , h , k , l , o , s , v , w and x / z .
they have been implicated in apoptosis regulation because of their cytoplasmic release due to increased lysosomal membrane permeabilization ( lmp ) .
although they depend on acidic ph for optimal activity , they seem to retain some of their catalytic activity at physiological ph . therefore , upon their release into the cytoplasm they can directly cleave caspases   or , as recently reported , pro- and anti - apoptotic members of the bcl-2 family or xiap .
unfortunately , in most studies , lmp was artificially induced by lysosomotropic agents such as leu - leu - ome ( l - leucyl - l - leucine methyl ester ) , and it has therefore remained unclear if cathepsin release indeed plays an initiating role in apoptosis signaling .
report that cells deficient in bax / bak , apoptosomal components or caspase-3/-7 are resistant to lmp in response to various apoptotic stimuli , indicating that cathepsin release and action occurs downstream of mitochondria .
moreover , since some cathepsin knock - out cells ( b , l but not d ) exhibited delayed but not blocked apoptosis , this lysosomal signaling pathway seems to amplify rather than initiate apoptosis .
this process could nevertheless be physiologically relevant because a lack of this amplifcation loop would lead to delayed cell death and/or inefficent removal of apoptotic bodies and may be the underlying cause of a lack of resolution of inflammation due to neutrophil accumulation in cathepsin d knock - outs , reduced keratinocyte apoptosis resulting in periodic hair loss in cathepsin l - deficient mice   and diminished liver damage upon tumor necrosis factor ( tnf)-alpha challenge in mice deficient of cathepsin b .
offered a possibility to stabilize lysosomes by delivering hsp70 ( heat - shock protein 70 ) via the endocytotic pathway . through its interaction with an endolysosomal phospholipid
this could be exploited to correct niemann - pick disease - associated pathology ( in which acid spingomyelinase is defective ) .
conversely , as tumor cells express high levels of lysosomal hsp70 , a reduction of this protein may sensitize these cells to lmp and cathepsin - mediated cell death .
it has remained enigmatic why cells , even in clonal populations , undergo apoptosis at different rates despite simultaneous addition of an apoptotic stimulus .
although caspase-3 activation occurs within minutes after cytochrome c release , the duration of events preceding cytochrome c release can vary from 1 to 24 hours .
a number of factors could contribute to this population variability , including differences in cell cycle stages , metabolic rates , the local environment of single cells or stochasticity in biochemical reactions triggered by apoptotic stimuli .
now , two recent papers reveal that the cell - cell variability of apoptosis times is due to the divergence of the molecular composition of the cells . using a novel single fluorophore reporter to measure caspase-3/-7 activities inside cells , bhola et al .  
report that the onset of proteolytic activity is tightly synchronized amongst the most related sister cells and lost over successive generations .
similiarly , spencer et al .   found that variability in the apoptotic phenotype arises from cell - to - cell differences in protein levels of caspases , caspase inhibitors , bcl-2 family members , and so on , that exist before trail ( tnf - related apoptosis - inducing ligand ) exposure .
a particular protein state of a single cell is then transmitted to the daughter cells , giving rise to transient non - genetic heritability in cell fate .
due to ongoing protein synthesis , the protein content of sister cells rapidly diverges so that soon these cells are no more similar to each other than pairs of cells chosen at random .
although we have learned much about the regulation of mammalian caspases , their ordering within signaling cascades and their specific roles in particular cell death regimens in vitro and in vivo , we do not know yet the full scope of action of the two effector caspases-7 and -6 .
is caspase-6 only an amplifier caspase or does it play a discrete role under particular stress responses ? what about the large number of caspase substrates that have recently been identified by mass spectrometry ? can they be validated in vivo ( i.e. , at physiologically relevant caspase concentrations of 1 nm in dying cells ) and which of them , if any , exert a functional significance in mediating the execution of apoptosis ?
another major task will be to determine if caspase-2 is only a checkpoint controller , or also an apoptotic caspase at specific cell cycle stages , for example , leading to mitotic catastrophe , a form of apoptosis that may depend on the molecular profile of the cell . in this respect
it will be exciting to continue the recent work on determining how the protein content of apoptosis signaling molecules sets the threshold for death induction in single cells , preferentially supported by systems biology approaches as shown by spencer et al . .
this will have major implications for future anti - cancer therapies as resistance to chemotherapy is most likely due to such cell - to - cell variability within a given tumor cell population .
another crucial aspect will certainly be the better detection of apoptotic cells in diseased tissues using specific fluorescent probes and the development of more drugs that activate the caspase-3 proenzyme .
in particular , a drug mimicking the caspase-3 mutant described by walters et al .   holds great promise to activate caspase-3 without proteolytic processing and eventually to kill even tumor cells expressing high xiap levels .
finally , we desperately need to understand the exact role of non - caspase proteases in cell death regulation .
the implication of calpains , serine proteases , cathepsins and other proteases in this process seems to be a relatively recent evolutionary response to the complexity of biological processes in humans .
thus , these enzymes will be promising targets for future therapeutic intervention - for example , to save neurons or immune cells from degeneration - irrespective of whether these proteases amplify caspase - dependent signaling or trigger alternative caspase - independent processes .
our future work will include the identification and molecular characterization of more apoptotic and pro - inflammatory serine proteases and the discovery of the signaling pathway leading from caspases to lmp and cathepsin release from lysosomes .
 undoubtedly , caspases are the major driving force for apoptosis execution and mechanisms of their activation and inhibition have been largely unveiled . 
 recent progress has been made with regard to the exact intracellular ordering of caspases , monitoring their activities in vivo and unveiling their substrate degradomes . moreover , non - caspase proteases seem to assist caspases in the completion of the death execution program . here 
 we will consider some very recent data dealing with these aspects . 
 we will also provide novel insights into the mechanisms that dictate apoptotic variability within a cell population . 
Introduction and context
Major recent advances
Redundancy, substrate specificity and amplification loops in caspase signaling
Monitoring caspases 
With a little help from your friends: non-caspase proteases assisting in cell death
Together with you I die: the molecular basis for synchronous dying
Future directions
Competing Interests