Non-apoptotic Functions Research Articles

Apoptosis: A barrier against cancer no more?

Apoptosis: A barrier against cancer no more?

Non-apoptotic function of apoptotic proteins in the development of Malpighian tubules of Drosophila melanogaster

Drosophila metamorphosis is characterized by the histolysis of larval structures by programmed cell death, which paves the way for the establishment of adult-specific structures under the influence of the steroid hormone ecdysone. Malpighian tubules function as an excretory system and are one of the larval structures that are not destroyed during metamorphosis and are carried over to adulthood. The pupal Malpighian tubules evade destruction in spite of expressing apoptotic proteins, Reaper, Hid, Grim, Dronc and Drice. Here we show that in the Malpighian tubules expression of apoptotic proteins commences right from embryonic development and continues throughout the larval stages. Overexpression of these proteins in the Malpighian tubules causes larval lethality resulting in malformed tubules. The number and regular organization of principal and stellate cells of Malpighian tubules is disturbed, in turn disrupting the physiological functioning of the tubules as well. Strikingly, the localization of beta-tubulin, F-actin and Disclarge (Dlg) is also disrupted. These results suggest that the apoptotic proteins could be having non-apoptotic function in the development of Malpighian tubules.

Nonapoptotic Function of BAD and BAX in Long-Term Depression of Synaptic Transmission

It has recently been found that caspases not only function in apoptosis, but are also crucial for nonapoptotic processes such as NMDA receptor-dependent long-term depression (LTD) of synaptic transmission. It remains unknown, however, how caspases are activated and how neurons escape death in LTD. Here we show that caspase-3 is activated by the BAD-BAX cascade for LTD induction. This cascade is required specifically for NMDA receptor-dependent LTD but not for mGluR-LTD, and its activation is sufficient to induce synaptic depression. In contrast to apoptosis, however, BAD is activated only moderately and transiently and BAX is not translocated to mitochondria, resulting in only modest caspase-3 activation. We further demonstrate that the intensity and duration of caspase-3 activation determine whether it leads to cell death or LTD, thus fine-tuning of caspase-3 activation is critical in distinguishing between these two pathways.

Abstract LB-128: The N-terminus of the ASPP2 tumor suppressor binds Ras and enhances Ras/Raf/MEK activation to promote oncogene-induced cellular senescence

Abstract The ASPP2 tumor suppressor is a pro-apoptotic member of a family of p53 binding proteins. It functions in part by selectively enhancing p53 transcriptional activation of pro-apoptotic target genes. Although the ASPP2 C-terminal domain binds the p53 core domain, little is known about the physical and functional interactions of other proteins to the highly structured ASPP2 N-terminus. Moreover, mounting evidence from our lab and others demonstrate that ASPP2 also mediates non-apoptotic p53-independent functions. However, the molecular mechanisms remain poorly understood. Previous reports have found that ASPP2 can localize to the cytoplasm and have identified a potential Ras-association domain in the ASPP2 N-terminus. Therefore, we wondered if novel ASPP2 functions could be mediated by ASPP2 N-terminal interaction with cytoplasmic Ras. Using co-immunoprecipitation and immunofluorescence microscopy, we identified ASPP2 in a complex with active Ras on the plasma membrane. Moreover, we also found that this interaction required the ASPP2 N-terminus since BBP, a naturally occurring alternatively spliced ASPP2 isoform lacking the N-terminus, showed markedly reduced ability to bind Ras. To determine if ASPP2 could modulate Ras function, we ectopically expressed ASPP2 in cells and found enhancement of Ras activation and subsequent stimulation of ERK phosphorylation. Conversely, ASPP2 knockdown by siRNA drastically reduced ERK phosphorylation and activation by EGF. Although it is oncogenic in tumor cells, constitutive Ras activation in normal cells leads to premature senescence in a Raf-MEK-ERK activation dependent manner. To explore if ASPP2 enhanced senescence, we knocked down ASPP2 expression with siRNA and found that senescence induced by RasV12 lentiviral infection in normal human fibroblasts was significantly attenuated. This was similar to the attenuation of senescence achieved by the MEK inhibitor PD98059. Together, our results demonstrate a novel function of the ASPP2 N-terminus in promoting cellular senescence via Ras-interaction and stimulation of Raf-MEK-ERK activity. These findings suggest a novel mechanism by which ASPP2-induced senescence may mediate its tumor suppression ability, and opens new avenues for investigation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-128. doi:10.1158/1538-7445.AM2011-LB-128

Abstract 3794: BAX maintains mitochondrial bioenergetics in colon cancer cells

Abstract The dual functionality of the tumor suppressor, BAX, is implied by the non-apoptotic functions of other pro-apoptotic BCL-2 family proteins. To explore this, the mitochondrial morphology and ATP producing capability in both BAX-containing and BAX-deficient HCT-116 colon cancer cells were examined. While BAX deficient cells maintain the same mitochondrial mass as their BAX containing counterparts, these mitochondria were visibly smaller, more compact, and unable to maintain an organized network. Oxygen consumption and ATP levels were also reduced in these cells, a phenomenon that was replicated upon treatment of BAX-containing cells with the uncoupling agent, FCCP. These results, coupled with a marked decrease in citrate synthase activity, suggested that cells lacking BAX have a deficiency in the ability to generate ATP through oxidative phosphorylation. Inhibition of endogenous BAX through treatment with small interfering RNAs reinforced the necessity of BAX activity in the maintenance of aerobic respiration and the production of ATP. An examination of the localization of full length and C-terminal truncated BAX in non-apoptotic, BAX deficient cells, indicated that a small fraction of the existing cytosolic BAX is needed at the mitochondrial membrane to maintain ATP production. However, this function is not dependent upon the C-terminal helix. Co-expression of BAX and BCL-2 resulted in a subsequent loss of ATP production, implying that, even under non-apoptotic conditions, an antagonistic interaction exists between the two proteins. These findings infer that a basal amount of BAX associated with the mitochondria is necessary to maintain mitochondrial bioenergetics. The discovery of this novel, non-apoptotic role for BAX in the maintenance of ATP synthesis and suggests that complete abrogation of BAX would be detrimental to cancer cells dependent on energy production via aerobic respiration. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3794. doi:10.1158/1538-7445.AM2011-3794

RET Modulates Cell Adhesion via Its Cleavage by Caspase in Sympathetic Neurons

RET is a tyrosine kinase receptor involved in numerous cellular mechanisms including proliferation, neuronal navigation, migration, and differentiation upon binding with glial cell derived neurotrophic factor family ligands. RET is an atypical tyrosine kinase receptor containing four cadherin domains in its extracellular part. Furthermore, it has been shown to act as a dependence receptor. Such a receptor is active in the absence of ligand, triggering apoptosis through a mechanism that requires receptor intracellular caspase cleavage. However, different data suggest that RET is not always associated with the cell death/survival balance but rather provides positional information. We demonstrate here that caspase cleavage of RET is involved in the regulation of adhesion in sympathetic neurons. The cleavage of RET generates an N-terminal truncated fragment that functions as a cadherin accessory protein, modifying cadherin environment and potentiating cadherin-mediated cell aggregation. Thus, the caspase cleavage of RET generates two RET fragments: one intracellular domain that can trigger cell death in apoptotic permissive settings, and one membrane-anchored ectodomain with cadherin accessory activity. We propose that this latter function may notably be important for the adequate development of the superior cervical ganglion.

Non-apoptotic Fas (CD95) signaling on T cells regulate the resolution of airway inflammation in a murine model of asthma (55.9)

Abstract Asthma is a Th2-mediated airway disease associated with chronic lung inflammation and mucus production. The mechanism by which inflammation resolves in the lung remains unidentified. We have previously shown that Fas deficiency on T cells in an adoptive transfer model of experimental asthma, results in a delay in resolution of inflammation compared with control. Surprisingly, using T cells with survival advantages independent of Fas on T cells (Bim-/- and BcLxL-tg), we found that inflammation resolution was not delayed, thus suggesting a dependency of T cells on a Fas-specific mechanism for normal resolution of inflammation. Fas has long been known to function as a death initiator by activating caspase-mediated apoptosis. However, recently it has also become clear that Fas has non-apoptotic functions including activation of NF-κB via MAPK pathways. Using an adoptive transfer model, Fas mutant T cells capable of non-apoptotic Fas signaling, but not apoptotic Fas signaling, were sufficient to produce normal resolution. Further, we have found that in vitro skewed Th2 cells that are resistant to Fas-mediated apoptosis, still signal through the non-apoptotic Fas pathway. Together these findings demonstrate that non-apoptotic Fas signaling, rather than apoptotic signaling, in T cells is important for normal resolution of airway inflammation in a murine model of experimental asthma.

BAX supports the mitochondrial network, promoting bioenergetics in nonapoptotic cells

The dual functionality of the tumor suppressor BAX is implied by the nonapoptotic functions of other members of the BCL-2 family. To explore this, mitochondrial metabolism was examined in BAX-deficient HCT-116 cells as well as primary hepatocytes from BAX-deficient mice. Although mitochondrial density and mitochondrial DNA content were the same in BAX-containing and BAX-deficient cells, MitoTracker staining patterns differed, suggesting the existence of BAX-dependent functional differences in mitochondrial physiology. Oxygen consumption and cellular ATP levels were reduced in BAX-deficient cells, while glycolysis was increased. These results suggested that cells lacking BAX have a deficiency in the ability to generate ATP through cellular respiration. This conclusion was supported by detection of reduced citrate synthase activity in BAX-deficient cells. In nonapoptotic cells, a portion of BAX associated with mitochondria and a sequestered, protease-resistant form was detected. Inhibition of BAX with small interfering RNAs reduced intracellular ATP content in BAX-containing cells. Expression of either full-length or COOH-terminal-truncated BAX in BAX-deficient cells rescued ATP synthesis and oxygen consumption and reduced glycolytic activity, suggesting that this metabolic function of BAX was not dependent upon its COOH-terminal helix. Expression of BCL-2 in BAX-containing cells resulted in a subsequent loss of ATP measured, implying that, even under nonapoptotic conditions, an antagonistic interaction exists between the two proteins. These findings infer that a basal amount of BAX is necessary to maintain energy production via aerobic respiration.

Expression of caspase‐8 and caspase‐9 in rat hippocampus during postnatal development

The role of caspases in the regulation of apoptosis of neurons during development is well established. An emerging body of evidence indicates that caspases may also play significant roles which are nonapoptotic. We have demonstrated previously that the executor caspase-3 exhibited a unique pattern of spatiotemporal expression in the postnatal rat hippocampal subregions, and the activation of caspase-3 in different hippocampal neurons appeared to have distinct roles during postnatal development. In the present study, we examined the expressions of initiator caspases in the hippocampus, using immunofluorescent staining for caspase-8 and caspase-9, and Hoechst 33342 staining for nuclear chromatin to assess caspase-8 and -9 expression in the CA1, CA3, and the dentate gyrus (DG) on postnatal days (P) 0, P2, P4, P7, P14, P21, P28, P56. The results indicate that caspase-8 and caspase-9 were expressed in pyramidal neurons of CA1 and CA3 fields, and granular neurons of the DG during development. Caspase-8 was expressed in a general upward trend while caspase-9 showed a slight downward pattern, but still remained at high levels in the adult hippocampus. The expression profiles of caspases-8 and -9 are distinct from that of the apoptotic cells. These data indicate that caspase-8 may be involved not only in the classical apoptotic function, but also in the cell death of necrosis, and in response to different insults and other nonapoptotic functions. Caspase-9 plays a role in apoptosis during postnatal development, but it may have other functions as well.

Cleaved caspase-3 expression after experimental stroke exhibits different phenotypes and is predominantly non-apoptotic

Cleaved caspase-3 (CC3) is well known as an executioner protease of apoptosis following brain ischemia. However, an increasing body of evidence suggests several non-apoptotic functions of CC3. To improve our understanding of the relation between cell death-related and non-adverse effects of postischemic caspase-3 activation, we examined the spatiotemporal distribution and identity of CC3-positive cells at days 2, 3 and 4 after permanent middle cerebral artery occlusion in rats. The lacking colocalization of CC3 and TUNEL staining indicated, that CC3 expression was predominantly non-apoptotic. Nuclear CC3 expression was frequently found to be colocalized with GFAP-positive astrocytes within the tissue adjacent to the infarct, whereas cytoplasmatic CC3 expression occurred solely in the lesion. Multiple fluorescence labeling revealed costaining of cytoplasmatic CC3 with markers directed against astrocytes, macrophages/microglia and supposedly pericytes. Our findings suggest that CC3 expression was predominantly associated with cellular responses to stroke such as reactive astrogliosis and the infiltration of macrophages.

Influence of FAS on murine mast cell maturation

FAS has been shown to be involved in the regulation of many immune processes by induction of cellular apoptosis. However, accumulated evidence shows that FAS signaling also exhibits nonapoptotic functions, such as induction of cell proliferation and differentiation. FAS is the only death receptor known to be expressed on murine mast cells (MCs). To evaluate the role of FAS on murine MC maturation. Mouse bone marrow-derived MCs (BMMCs) or peritoneal MCs were derived from FAS-deficient, FASlpr/lpr, and congenic wild-type strains. The MC degranulation and cytokine release after IgE activation was assessed by measuring β-hexosaminidase, interleukin 13, and tumor necrosis factor α release. Transmission electron microscopy analysis was performed to evaluate the level of BMMC maturation. The surface markers and intracellular preformed mediators were measured as well. Our data reveal that FAS deficiency has an impact on IgE-dependent activation of BMMCs, resulting in a significant decrease in β-hexosaminidase, interleukin 13, and tumor necrosis factor α release. The total content of preformed mediators (eg, tryptase and β-hexosaminidase) was reduced in BMMCs derived from FAS-deficient mice. We also found that the level of FcεRI in peritoneal mast cells from FAS-deficient mice was significantly diminished. FAS deficiency also influenced the kinetics of BMMC maturation as was revealed by transmission electron microscopy analysis. Our data show that FAS has an impact on the regulation of mouse MC maturation in vitro.

Stimulation of Fas (CD95) induces production of pro-inflammatory mediators through ERK/JNK-dependent activation of NF-κB in THP-1 cells

Although Fas is known to be an apoptosis triggering molecule, accumulating studies indicate that Fas has non-apoptotic functions in certain cases. In an effort to identify the role of Fas in macrophage function, the human macrophage-like cell line THP-1 was analyzed after treatment with agonistic anti-Fas monoclonal antibody or co-incubation with FasL-expressing cells. Stimulation of Fas induced the expression of pro-inflammatory mediators such as matrix metalloproteinase (MMP)-9 and IL-8. The specificity of the reaction was confirmed by the transfection of Fas-specific siRNAs which resulted in a suppression of Fas expression as well as the responsiveness to the agonistic antibody. Utilization of various signaling inhibitors and ELISA-based NF-κB DNA binding assay demonstrated that the signaling initiated from Fas is mediated by mitogen activated protein kinases (MAPKs) including extracellular-signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) which induce subsequent activation of NF-κB. Furthermore, mixed cell culture experiment demonstrated that Fas can be activated through interaction with membrane-bound form of FasL during cell-to-cell interaction. These data indicate that Fas plays a role as an activation inducing molecule through interaction with its counterpart and Fas-mediate events are mediated by ERK/JNK MAPKs which subsequently activate NF-κB for the transcriptional activation of pro-inflammatory mediators.

Apoptotic and Non-apoptotic Caspase Functions in Neural Development

During neural development, massive cell death occurs in both vertebrates and invertebrates. Caspase is a central player in apoptosis that is evolutionally conserved. Genetic manipulation of the caspase activity in Drosophila and mice has revealed that caspases control cell fate through apoptotic and non-apoptotic mechanisms, to ensure appropriate cell differentiation and maturation in the developing nervous system.

Bid regulates the immunological profile of murine microglia and macrophages

Apoptosis is a controlled cell-death process mediated inter alia by proteins of the Bcl-2 family. Some proteins previously shown to promote the apoptotic process were found to have nonapoptotic functions as well. Microglia, the resident immune cells of the central nervous system, respond to brain derangements by becoming activated to contend with the brain damage. Activated microglia can also undergo activation-induced cell death. Previous studies have addressed the role of core apoptotic proteins in the death process, but whether these proteins also play a role or not in the activation process is not been reported. Here we explore the effect of the BH3-only protein Bid on the immunological features of microglia and macrophages. Our results showed that Bid regulates both the phagocytotic activities and the inflammatory profiles of these cells. Deficiency of Bid attenuated the phagocytotic activity of primary microglia and peritoneal macrophages. It also changed the expression profile of distinct inflammation-related genes in lipopolysaccharide-activated microglia and peritoneal macrophages in vitro and in an in vivo sepsis-like paradigm. Notably, similar changes followed downregulation of Bid in the N9 microglial cell line. Cell death could not be detected in any of the systems examined. Our findings demonstrate that Bid can regulate the immunological profiles of activated microglial and macrophages, via a novel nonapoptotic activity. In view of the critical role of these cells in various pathologies, including acute and chronic brain insults, our findings suggest that impairments in Bid expression may contribute to these pathologies also via a nonapoptotic activity.

Impact of death receptor signaling on the malignancy of pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma, PDAC, is a type of tumor with late diagnosis, rapid progression and poor prognosis. It is mostly unresponsive to chemo- and radiation therapy due to a marked resistance against apoptosis induction via both the intrinsic and extrinsic pathway. Accordingly, also activation of death receptors of the TNF-receptor superfamily does not or hardly induces apoptosis in PDAC cells. The reasons for this resistance include expression of multiple inhibitory proteins that block apoptotic signaling at almost every step of the signaling cascade. Moreover and more importantly, death receptors such as CD95/Fas, TRAIL-R1/-R2 and TNF-R1 exhibit marked non-apoptotic functions after ligand binding leading to strong proinflammatory responses, which contribute to survival, proliferation, migration and invasion, causing an even more pronounced malignant phenotype. This non-apoptotic signal transduction is facilitated via distinct adapter proteins, such as TRAF2 that bind to death receptor complexes and stimulate proinflammatory signal pathways leading to activation of NF-κB and AP-1 transcription factors. Therapeutic interventions in oncology utilizing death ligands, e.g. TRAIL, or triggering death receptors indirectly, must be carefully evaluated with respect to possible “side effects” and should be designed in a way counteracting non-apoptotic proinflammatory signaling.

The Enigmatic Roles of Caspases in Tumor Development

One function ascribed to apoptosis is the suicidal destruction of potentially harmful cells, such as cancerous cells. Hence, their growth depends on evasion of apoptosis, which is considered as one of the hallmarks of cancer. Apoptosis is ultimately carried out by the sequential activation of initiator and executioner caspases, which constitute a family of intracellular proteases involved in dismantling the cell in an ordered fashion. In cancer, therefore, one would anticipate caspases to be frequently rendered inactive, either by gene silencing or by somatic mutations. From clinical data, however, there is little evidence that caspase genes are impaired in cancer. Executioner caspases have only rarely been found mutated or silenced, and also initiator caspases are only affected in particular types of cancer. There is experimental evidence from transgenic mice that certain initiator caspases, such as caspase-8 and -2, might act as tumor suppressors. Loss of the initiator caspase of the intrinsic apoptotic pathway, caspase-9, however, did not promote cellular transformation. These data seem to question a general tumor-suppressive role of caspases. We discuss several possible ways how tumor cells might evade the need for alterations of caspase genes. First, alternative splicing in tumor cells might generate caspase variants that counteract apoptosis. Second, in tumor cells caspases might be kept in check by cellular caspase inhibitors such as c-FLIP or XIAP. Third, pathways upstream of caspase activation might be disrupted in tumor cells. Finally, caspase-independent cell death mechanisms might abrogate the selection pressure for caspase inactivation during tumor development. These scenarios, however, are hardly compatible with the considerable frequency of spontaneous apoptosis occurring in several cancer types. Therefore, alternative concepts might come into play, such as compensatory proliferation. Herein, apoptosis and/or non-apoptotic functions of caspases may even promote tumor development. Moreover, experimental evidence suggests that caspases might play non-apoptotic roles in processes that are crucial for tumorigenesis, such as cell proliferation, migration, or invasion. We thus propose a model wherein caspases are preserved in tumor cells due to their functional contributions to development and progression of tumors.

Activation and Specificity of Human Caspase-10

Two apical caspases, caspase-8 and -10, are involved in the extrinsic death receptor pathway in humans, but it is mainly caspase-8 in its apoptotic and nonapoptotic functions that has been an intense research focus. In this study we concentrate on caspase-10, its mechanism of activation, and the role of the intersubunit cleavage. Our data obtained through in vitro dimerization assays strongly suggest that caspase-10 follows the proximity-induced dimerization model for apical caspases. Furthermore, we compare the specificity and activity of the wild-type protease with a mutant incapable of autoprocessing by using positional scanning substrate analysis and cleavage of natural protein substrates. These experiments reveal a striking difference between the wild type and the mutant, leading us to hypothesize that the single chain enzyme has restricted activity on most proteins but high activity on the proapoptotic protein Bid, potentially supporting a prodeath role for both cleaved and uncleaved caspase-10.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) induces chemotactic migration of monocytes via a death receptor 4-mediated RhoGTPase pathway

This study tested the hypothesis that TRAIL could play a role in regulating monocyte migration. TRAIL has been widely studied for its anti-tumor function and signaling mechanisms. Using chemotaxis and mouse air-pouch model analyses, we determined that TRAIL-induced chemotactic migration of THP-1 human leukemia and LPS-primed primary human monocytes as well as LPS-stimulated BALB/c mouse monocytes in vivo. To expand the understanding of the TRAIL signaling pathway in this process, we found that the TRAIL receptor DR4 was highly expressed in THP-1 and LPS-primed primary monocytes but not in the non-primed primary monocytes. DR4 neutralization antibody specifically suppressed TRAIL-induced migration of the monocytes. Furthermore, PI3K, Rho GTPase and its downstream effectors, MLC and Pak1, were activated during cell migration. PI3K inhibitors and dominant negative mutants of RhoGTPase blocked monocyte migration toward TRAIL, indicating that PI3K and RhoGTPases were involved in the migration signaling. The DR4 neutralization antibody blocked the activation of PI3K and Rho GTPase effectors in the cells. Thus, these data support the hypothesis that TRAIL induces monocyte migration mediated by TRAIL receptor DR4 via the RhoGTPase signaling pathway. This study is expected to provide novel evidence of the non-apoptotic function of TRAIL in immune defense.

Enzymatically active single chain caspase-8 maintains T-cell survival during clonal expansion

The extrinsic, or death receptor, pathway integrates apoptotic signals through the protease caspase-8 (casp8). Beyond cell death regulation, non-apoptotic functions of casp8 include its essential requirement for hematopoiesis and lymphocyte clonal expansion, and tempering of autophagy in T cells. However, the mechanistic basis for the control of these disparate cellular processes remains elusive. Here, we show that casp8-deficient T-cell survival was rescued by enzymatically active, but not inactive, casp8-expressing retroviruses. The casp8 catalytic induction in proliferating T cell occurred independent of extrinsic and intrinsic apoptotic-signaling cascades and did not induce casp8 proteolytic processing. Using a biotinylated probe selectively targeting enzymatically active caspases, catalytically active full-length casp8 was found in vivo in dividing T cells. A casp8 D387A processing mutant was able to rescue casp8-deficient T-cell proliferation, validating that casp8 self-processing is not required for its non-apoptotic function(s). Finally, casp8 activity was highest in CD8(+) T cells, the most rapidly proliferating subset. These results show that the catalytically competent form of casp8 is required for rapid T-cell proliferation in response to TCR ligation, but that processing of the caspase is only necessary to promote apoptosis.

Functional dissection of the granzyme family: cell death and inflammation

Cytotoxic lymphocytes rapidly respond and destroy both malignant cells and cells infected with intracellular pathogens. One mechanism, known as granule exocytosis, employs the secretory granules of these lymphocytes. These include the pore-forming protein perforin (pfp) and a family of serine proteases known as granzymes that cleave and activate effector molecules within the target cell. Over the past two decades, the study of granzymes has largely focused on the ability of these serine proteases to induce cell death. More recently, sophisticated mouse models of disease coupled with gene-targeted mice have allowed investigators to ask why granzyme subfamilies are encoded on different chromosomal loci and what broader role these enzymes might play in inflammation and immune response. Here, we provide a brief overview of the granzyme superfamily, their relationship to pfp, and their reported functions in apoptosis. This overview is followed by a comprehensive analysis of the less characterized and developing field regarding the non-apoptotic functions of granzymes.