mitochondrial transmembrane potential apoptosis - inducing factor apoptotic protease - activating factor 1 bcl2-associated x protein b - cell cll / lymphoma 2 bh3 interacting domain death agonist dna - dependent protein kinase electron transport chain high temperature requirement protein a2 inhibitor of apoptosis inhibitor of caspase - activated dnase mitochondrial outer membrane permeabilization nicotinamide adenine dinucleotide nadh dehydrogenase ( ubiquinone ) 1 alpha subcomplex , 9 nadh dehydrogenase ( ubiquinone ) fe - s protein 1/2 nadh dehydrogenase ( ubiquinone ) flavoprotein 1 nicotinamide adenine dinucleotide phosphate ( nadph)-oxidase nuclear mitotic apparatus protein 1 12 , 17 and 20 kda cleavage fragments of the maturation process of caspase 3 poly-(adp - ribose ) polymerase reactive oxygen species
human cytotoxic lymphocytes ( ctls ) eradicate pathogen - infected or transformed cells primarily through the cytotoxic granule pathway , which relies on perforin ( pfn)- and granulysin - mediated delivery of the granzyme serine proteases into eukaryotic target cells or bacteria , respectively . among the 5 human granzymes ( a , b , h , k , and m ) and 10 mouse homologues ( a , b , c , g , e , f , g , k , m , and n ) , granzymes b and a ( gb and ga ) are the most abundantly expressed , and the best characterized . like caspases , gb cleaves its substrates after aspartic acid residues to induce cell death .
in fact , gb behaves as a hybrid between initiator and effector caspases  like initiator caspases , gb activates the effector caspases 3 and 7 and the proapoptotic bh3-only protein bid ; however , gb can also directly cleave important effector caspase substrates such as icad , parp-1 , lamin b , numa , dna - pkc and tubulin for cell death induction .
mitochondria adjust both their morphology and function , acting as hubs that integrate cues for the appropriate cellular response . in response to activation of death receptors , dna damage , or er stress , activation of bh3-only proteins of the bcl2 family
triggers the oligomerization of bax and bak leading to mitochondrial outer membrane permeabilization ( momp ) .
this critical step of many cell death pathways results in release of the apoptogenic factors cytochrome c ( cyt c ) , endog , smac / diablo , htra2 and aif from the mitochondria into the cytosol . as a consequence
, cytosolic cyt c triggers activation of apaf1 and caspase 9 apoptosome platform , and smac removes the repression of the inhibitor of apoptosis ( iap ) proteins .
, momp was considered sufficient for apoptogenic factor release ; however , recent findings suggest that the story is more complex .
in fact , reactive oxygen species ( ros ) are necessary for cyt c release .
cyt c engages in both electrostatic and hydrophobic interactions with inner mitochondrial membrane cardiolipins that are disrupted by ros for optimal release through momp .
gb triggers optimal cyt c , endo g , and smac release in a ros - dependent manner ( fig .
it is likely that , similar to cyt c , ros - mediated untethering of endo g and smac is required for their release through the bid / bax / bak - mediated momp in the gb pathway .
ros are also necessary for gb - induced dna fragmentation since ros scavengers significantly reduce and delay oligonucleosomal dna fragmentation without affecting icad cleavage and activation of cad , the endonuclease involved in this process ( fig .
this result could be predicted by the compromised release of endog from mitochondria in the absence of ros .
furthermore , ros were found to be necessary for the nuclear shuttling of endog during cisplatin - induced death of head and neck squamous carcinoma cells , suggesting that ros may also be required for endog and cad nuclear translocation following gb and pfn treatment ( fig .
an additional contribution of ros in apoptosis progression was revealed by the work of christoph borner 's group , who showed that during tnf apoptotic signaling , caspase 9 induced lysosomal membrane permeabilization ( lmp ) in a ros - dependent manner , leading to cathepsin release and further caspase activation and cell death .
overall , these findings are consistent with the inhibitory effect of ros scavengers on ctl - mediated cell death , implying that ros are active effectors of cell death . 

gb can directly cleave parp1 , lamin b , tubulin , numa , dna - pk , and icad to induce caspase - independent cell death .
gb also enters the mitochondria by an unknown mechanism and cleaves ndufs1 , ndufs2 , and ndufv1 from complex i to trigger ros production .
together with ros , momp allows the release of cyt c , endog , smac , htra2 , and aif ( the latter 2 are not represented here ) .
both cad and endog shuttle to the nucleus probably in a ros - dependent mechanism to induce oligonucleosomal dna fragmentation . ros and caspase 9
cooperate to trigger lysosomal membrane permeabilization and cathepsin release , thus amplifying caspase 3 activation . among other substrates ,
activated caspase 3 cleaves parp1 , lamin b , tubulin , numa , dna - pk , and icad , which together contribute to cell death . 
 
gb can directly cleave parp1 , lamin b , tubulin , numa , dna - pk , and icad to induce caspase - independent cell death .
gb also enters the mitochondria by an unknown mechanism and cleaves ndufs1 , ndufs2 , and ndufv1 from complex i to trigger ros production .
together with ros , momp allows the release of cyt c , endog , smac , htra2 , and aif ( the latter 2 are not represented here ) .
both cad and endog shuttle to the nucleus probably in a ros - dependent mechanism to induce oligonucleosomal dna fragmentation .
ros and caspase 9 cooperate to trigger lysosomal membrane permeabilization and cathepsin release , thus amplifying caspase 3 activation . among other substrates ,
activated caspase 3 cleaves parp1 , lamin b , tubulin , numa , dna - pk , and icad , which together contribute to cell death .
the production of ros during cell death has long been recognized ; however , a molecular mechanism for their production has only recently begun to be unraveled .
although one report suggested an extra - mitochondrial origin of ros during apoptosis , douglas green 's group first showed that staurosporine - mediated ros - dependent apoptosis requires caspase 3 cleavage of ndufs1 , the 75-kda subunit of the mitochondrial electron transport chain ( etc ) complex i. later , judy lieberman 's group reported that ga triggered a caspase- and momp - independent cell death that also required the disruption of complex i through the cleavage of ndufs3 to induce ros - dependent apoptosis .
interestingly , gb cleaves ndufs1 , ndufs2 , and ndufv1 subunits of this same complex to trigger ros - dependent apoptosis ( fig .
1 ) . actually , gb also alters complex ii and complex iii function , disrupts the supercomplex organization of the respiratory chain , and triggers a loss of the mitochondria cristae junctions . in this pathway ,
electron flow is required for ros production since 0 cells that carry a non - functional respiratory chain are resistant to granzyme - mediated ros production and cell death .
it is possible that cleavage of these complex i subunits exposes the electrons within the fe - s cluster to ambient oxygen for ros production .
additionally , the mitochondria targeted antioxidant mitoq completely abrogates gb - induced ros and cell death , further supporting a mitochondrial origin of ros in this context .
integration of these data indicates that 3 cell death pathways  caspase 3 , ga , and gb  converge on complex i for ros production during apoptosis .
i by ga and gb following human killer cell attack has been implicated in ros - dependent bacterial death , suggesting that cleavage of complex i subunits is an evolutionarily conserved mechanism of ros - dependent death .
our results indicate that ga and gb , which lack a mitochondrial targeting signal , still find their way into this double - membrane organelle to disrupt the respiratory chain and trigger an increase in ros .
we have also found that granzyme mitochondrial entry is momp - independent but requires an intact membrane potential .
do ros act through non - specific systemic redox - modification of macromolecules or through a discrete set of targets ?
how many ros are needed ? which radical species are most effective for the various contributions of ros to cell death ?
putting emphasis on redox signaling during apoptosis will undoubtedly open new windows of investigation and allow the characterization of useful biomarkers of cell death .
jerome thiery ( gustave roussy cancer campus , villejuif , france ) and dr michael walch ( university of fribourg , switzerland ) for proofreading this manuscript .
this work was supported by ambizione grant snsf pz00p3_126710/1 , erc starting grant erc-2010-stg_20091118 and subsides , from the carlos and elsie de reuter foundation and schmidheiny foundation .
 reactive oxygen species ( ros ) are involved in cell signaling , aging , and death and play a role in carcinogenesis . however , whether ros are bystanders or active effectors of apoptosis was unclear until recently . 
 new evidence shows that the killer lymphocyte protease granzyme b activates a conserved biochemical pathway centered on respiratory chain disruption to trigger mitocentric ros - dependent apoptosis . 