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Biochimica et Biophysica Acta 1773 (2007) 1491–1499 www.elsevier.com/locate/bbamcr

Minireview A more serine way to die: Defining the characteristics of -mediated cell death cascades ⁎ A.R. O’Connell, C. Stenson-Cox

National Centre for Biomedical and Engineering Science, National University of Ireland, Galway, Ireland Received 23 February 2007; received in revised form 11 July 2007; accepted 1 August 2007 Available online 14 August 2007

Abstract

The morphological features observed by Kerr, Wylie and Currie in 1972 define apoptosis, necrosis and autophagy. An appreciable number of alternative systems do not fall neatly under these categories, warranting a review of alternative proteolytic machinery and its contribution to cell death. This review aims to pinpoint key molecular features of serine protease-mediated pro-apoptotic signalling. The profile created will contribute to a standard set of biochemical criteria that can serve in differentiating within cell death subtypes. © 2007 Elsevier B.V. All rights reserved.

Keywords: Apoptosis; Serine protease; Caspase; Mitochondria

1. Introduction ability (MOMP) triggering the release of apoptogenic factors. Although the underlying mechanism of MOMP induction has The knowledge that cell death is an essential event in the life not been fully ascertained it is known to be regulated by of multi-cellular organisms has been around for more than members of the Bcl-2 family (Fig. 11). Anti-apoptotic Bcl-2 and 150 years. In 1972 Kerr et al., coined the term ‘apoptosis’ to Bcl-xl proteins block MOMP, whilst pro-apoptotic BH-3 describe a distinct morphological pattern of physiologically domain only proteins; Bim and Bid promote MOMP through occurring cell death [1]. More recently, an increasing number of activation of multi-domain proteins Bax and Bak [2,5,6]. ‘alternative’ cell death systems are being described that do not Apoptogenic factors released include cytochrome c (cytC), strictly conform to accepted apoptotic criteria, differing largely apoptosis inducing factor (AIF), SMAC (Second mitochondria- in terms of the proteolytic machinery employed by the dying derived activator of caspase)/DIABLO (Direct IAP-Binding cell. We are particularly interested in the roles of serine proteases Protein with Low pI) and the serine protease Omi/HtrA2 [5]. The in that regard. role of cytC in cell death is well established, contributing to caspase-9 activation through the formation of a cytosolic 2. Apoptosis apoptosome complex and the subsequent activation of cas- pase-3 (Fig. 12) [7]. Caspase-3 targets a wide array of important Apoptosis is a carefully regulated process wherein there structural and regulatory proteins for cleavage such as DFF45 exists an absolute requirement for controlled proteolytic (DNA fragmentation factor-45)/ICAD (inhibitor of caspase- activation [2]. Almost since their discovery, activation of cys- activated DNase) and PARP (poly(ADP-ribose)polymerase) teinyl aspartate-specific proteases, the caspases, has been (Fig. 13). [8]. The extrinsic pathway is triggered by ligation of considered synonymous with pro-apoptotic signalling [3,4]. death receptors such as FAS/CD95 and TNF to natural ligands Caspases are sequentially activated by two main mechan- present in the extracellular milieu. Subsequent recruitment of isms. In the intrinsic pathway, cytotoxic drugs or irradiation, for pro-caspase-8 to the receptor via adaptor molecules autoproteo- example, can activate mitochondrial outer membrane perme- lytically activates caspase-8 that then cleaves caspase-3 directly (Fig. 14) or indirectly through the activation of the intrinsic 5 ⁎ Corresponding author. Tel.: +353 91 495207; fax: +353 91 494596. pathway (Fig. 1 ) [2]. In the latter, caspase-8 cleaves the pro- E-mail address: [email protected] (C. Stenson-Cox). apoptotic BH3-only Bcl-2 family member, Bid which then

0167-4889/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.bbamcr.2007.08.002 1492 A.R. O’Connell, C. Stenson-Cox / Biochimica et Biophysica Acta 1773 (2007) 1491–1499 translocates to the mitochondria and induces cytC release. the pro-caspases-7, and -8 results in their inactivation and Activation of either pathway ultimately results in the ordered negatively impacts upon apoptotic events [19] whereas cleavage dismantling and demise of the cell. of pro-caspase-9, -12 and Bcl-xl promotes apoptosis [19,20]. However, caspase inhibition does not always prevent cell Calpain-mediated Bid cleavage has been demonstrated as an death [9,10] and their targeted disruption does not always impair essential early caspase-independent event during cisplatin- apoptosis [11], therefore, roles for non-caspase proteases such as induced cell death in human melanoma cells, characterising the calpains, and serine proteases in cell death have one of two activated cell death pathways; the other involving recently attracted more interest [12,13]. Activation of calcium- Bak modulation [21]. Cathepsins which are cysteine proteases dependent calpain family members has been described in from the lysosomal compartment have also been implicated in a caspase-independent cell death, autophagy and necrosis number of cell death processes [22]. Ligation of death receptors [14,15]. The ubiquitously expressed μ and m-calpains promote like the TNFα (Tumour Necrosis Factor-α) receptor can induce both apoptotic and survival signals in a cell and stimuli-specific the release of B into the cell cytosol leading to the manner [15–17]. Calpains activated in response to the activation of caspase-dependent and independent cell death accumulation of cytosolic calcium ions leads to the cleavage [22–24] and cathepsins D and B have been implicated in of a number of substrates, including α-spectrin [18]. Cleavage of camptothecin-induced death of liver cancer cells [25].

Fig. 1. Schematic illustrating the points of involvement of serine proteases in the apoptotic cascade. ΔΨm indicates change in dissipation of the mitochondrial inner transmembrane potential that can occur before, during or after MOMP. Numbers refer to pathways discussed in text and correlate to the superscript numbers assigned there. Serine proteases are highlighted in yellow. Where the molecular weight of the serine protease involved is known, it is given (e.g. p16) where not the protease is denoted as pX. Endonucleases are outlined in red; MnEndo, manganese-dependent endonuclease; EndoX, yet to be identified endonuclease. A.R. O’Connell, C. Stenson-Cox / Biochimica et Biophysica Acta 1773 (2007) 1491–1499 1493

Cathepsins are released into the cytosol prior to mitochondrial integrity largely through proteolytic inactivation of DFF45/ membrane-potential changes [26,27] and can activate caspases ICAD that acts as an endogenous inhibitor of a DNase (DFF40/ indirectly through cleavage of Bid [27,28]. CAD) (Fig. 13) [41]. Gr B has been shown to process DFF45 The involvement of serine proteases within the apoptotic in vitro and in vivo at the caspase-3 cleavage site, and to machinery is the least well understood of the non-caspase mediate DNA fragmentation in the absence of caspase-3 proteases and only a limited number of pro-apoptotic serine activity (Fig. 110) [42,43]. proteases have been isolated. This is in contrast to the large The relative importance of the direct caspase pathway com- amount of data expounding their contribution to various pared to the Bid pathway and cleavage of other pro-apoptotic apoptotic events. It is evident that the established apoptosis- substrates during CL-mediated killing has been the subject of associated serine proteases cannot be responsible for all of the much debate. Central to this has been the assumption that mouse potential serine protease-mediated functions observed to date. and human Gr B possess identical substrate specificities and as a This implies that there is much more to learn regarding the result experiments have used Gr B from either human or murine influence and identity of serine proteases within apoptosis. In sources inter-changeably across species. However, it has now this review we summarise the current data pertaining to serine been clearly demonstrated that mouse and human Gr B exhibit proteases in the initiation and execution phases of apoptosis. distinct specificity and different abilities to engage with the caspase or Bid apoptotic machinery [35,44,45]. Whereas both 3. and cell death human and mouse Gr B process pro-caspase-3, cleaving species- specific caspase more efficiently than unmatched substrate, Granzymes are serine proteases found exclusively in the mouse Gr B fails to cleave Bid, pro-caspase-8 and ICAD, even granules of activated cytotoxic T lymphocytes (CTL) and when the human equivalent does so efficiently under the same natural killer (NK) cells and play a critical role in eliminating experimental conditions suggesting that Bid is not a critical virally infected and malignant cells [29,30]. Cytotoxic granules mediator of mouse Gr B-mediated cytotoxicity. Complimenting also contain a pore-forming protein, perforin, which upon these studies are the observations that serpins prevent - recognition and contact with target cells, mediate the delivery of induced death in a species-specific manner and that human and the granzymes into the intermembrane space of the target cells mouse Gr B serpins are not evolutionarily conserved [45,46]. [31]. and B are the two most abundant and extensively studied members of the granzyme family and studies 5. Granzyme A in cytotoxic lymphocytes (CL) from knockout mice have confirmed that they independently and synergistically induce Granzyme A (Gr A) is a highly selective tryptic protease that cell death through distinct apoptotic pathways [32]. triggers a rapid form of cell death that exhibits all of the mor- phological features associated with apoptosis: membrane 4. Granzyme B blebbing, chromatin condensation and nuclear fragmentation [47]. However, Gr A does not activate caspase family members, Granzyme B (Gr B) is a 32-kDa protease that cleaves sub- caspase inhibition fails to prevent Gr A-induced death and Bcl-2 strates following an aspartate residue, thus exhibiting substrate overexpression does not inhibit Gr A cytolysis [47,48].In specificity similar to that of caspase family members. It is well addition Gr A does not cleave many key caspase targets established that Gr B can engage multiple components of the including lamin B and PARP [47]. During cell death Gr A apoptotic machinery in target cells triggering direct and indirect rapidly accumulates in the nucleus, where it appears to target the activation of caspases and the cleavage of a number of pro- nuclear envelope protein lamin, the chromatin structural protein apoptotic proteins (Fig. 18) [29–32]. Gr B can directly process Histone H1 and DNA [49–52]. Indeed, DNA damage is a and activate pro-caspases-3 and -7 and can induce a partial defining characteristic of the caspase-independent Gr A cell cleavage of caspase-8 and -10 [33–35]. The activated caspase-3 death pathway, marked by single-stranded nicks resulting in can then process pro-caspase-9, -2, and -6 or complete the Gr large DNA fragments [52]. A 270–440 kDa multi-protein B-initiated maturation of caspase-8 and -10 [34]. Gr B also complex, known as the SET complex, has recently been triggers indirect caspase activation through cleavage of the identified as being responsible for Gr A-mediated DNA damage BH3-domain only protein Bid leading to MOMP initiation and [53,54]. Nucleus translocation of the SET complex from the cytochrome c release [36–38]. Cells deficient in Bid are cytosol is an early event in GrA-mediated cell death. The SET resistant to Gr B-induced apoptosis, and continue to proliferate complex is comprised of three GrA substrates-the nucleosome in long-term survival assays (Fig. 17) [38]. More recently, the assembly protein SET (putative HLA-associated protein II, PP2A Bcl-2 family member Mcl-1 has also been identified as a Gr B template-activating factor (TAF)-1, or I2 ), the DNA bending substrate that when cleaved allows the release of Bim and protein HMG-2, and the base excision repair endonuclease induction of MOMP [39,40]. Gr B can also induce features Ape1. Cleavage of these substrates by GrA destroys all of their associated with apoptosis through direct cleavage of specific known functions [55,56]. The complex also contains a 25-kDa proteins. For instance, Gr B mediates cleavage of the Rho- Mg2+-dependent DNase; NM23-H1 (GzmA-activated DNase) regulated kinase Rock II, a regulator of actomyosin contraction or GAAD, whose activity is normally held in check by its that results in membrane blebbing and Gr B shares the caspase- inhibitor SET (IGAAD). When SET is cleaved it allows the 3 substrate; DFF45/ICAD [30]. Caspase-3 mediates genomic endonuclease, together with an exonuclease TREX1 that binds 1494 A.R. O’Connell, C. Stenson-Cox / Biochimica et Biophysica Acta 1773 (2007) 1491–1499 to SET, to make the characteristic DNA nicks [57]. In terms of gous deletion of the Prss2 [63]. The appearance of these regulation, at the level of the mitochondrion, proteolytically animals is akin to the mnd2 mice, characterised by a active Gr A triggers a rapid and sustained increase of Parkinsonian-like phenotype and early death resulting from the intracellular reactive oxygen species (ROS) and a concomitant loss of striatal neurons. The similarity between knockout loss of transmembrane potential that continues in the presence of animals that lack all expression of Omi to that of the mnd2 caspase inhibition and Bcl-2 overexpression [48]. Gr A does not mouse, exhibiting loss of protease activity only, raises questions cleave Bid and despite the loss of transmembrane potential, the about the physiological significance of Omi in apoptosis outer mitochondrial membrane remains intact preventing the regulation. Omi's IAP binding appears to be a highly redundant release of apoptogenic factors such as cytochrome c, AIF, function, at least in mice, for which mediation by other proteins EndoG. Although the exact molecular mechanisms responsible is necessary. for Gr A-mediated mitochondrial damage are undetermined, the affects are direct, associated with opening of the PT pore, and are 7. Apoptotic protein 24 (AP24) neither cell-type nor species specific. Functionally Gr A-induced ROS accumulation has been shown to trigger the translocation of AP24 is a 24-kDa serine protease with an -like the SET complex, from the cytoplasm to the nucleus [47,48]. activity that triggers oligonucleosomal DNA fragmentation [64]. The mechanism by which this occurs remains to be resolved. This activity has been observed in the cytosol and nucleus of tumour cell lines undergoing apoptosis, from which it has been 6. Omi and cell death partially purified [65]. Activation of AP24 occurs in response to a variety of stimuli including TNF, UV irradiation and Omi is a mammalian serine protease belonging to the HtrA chemotherapeutic agents [66,67]. Inhibition of protein synthesis (high temperature requirement A) protease/chaperone family, does not affect AP24 activation indicating that the protein is members of which are essential in the survival of high tem- expressed constitutively in either an active sequestered, or an peratures or oxidative stress. Omi can be catalytically processed inactive form, prior to exposure to an apoptotic stimulus [67]. during apoptosis and released from the mitochondria whereupon AP24 induces DNA fragmentation indirectly, by inactivating the it binds to IAPs neutralizing their inhibitory action upon serpin, leukocyte elastase inhibitor (LEI), through translational caspases (Fig. 16) [58]. Omi also possesses a caspase- modification and converting it to an endonuclease (L-DNase II) independent pro-apoptotic serine protease activity (Fig. 16); [64,68]. The resultant DNase II translocates to the nucleus and deletion of a tryspin-like domain results in partial inhibition of subsequently induces DNA degradation. Regulators of AP24 apoptosis [59] and cell death induced by Omi overexpression activation include sphingomyelin and calcium/calmodulin- occurs under caspase inhibiting conditions and in APAF-1 and dependent kinase II (CaMKII) and overexpression of Bcl-2 caspase-9 null cells [60,61]. inhibits AP24 activity in HL-60 cells [65,69–71]. Omi's relevance in cell death has been called into question however, with data from recent gene knockout studies and the 8. -type (uPA) mnd2 (motor neuron degeneration 2) mouse implying that although Omi can bind IAPs, it does not play a role in promoting The urokinase plasminogen activator (uPA) is a 53-kDa cell death, but rather acts as a protease to remove misfolded -like serine protease which converts the zymogen proteins in the mitochondria. Studies in the mnd2 mouse, a plasminogen into active , a broad spectrum protease disease characterised by altered gait, muscle wasting and early [72]. uPA is secreted as a pro-form, and its activation is mediated death, has shown that Omi can exert cell survival and death. In by its receptor uPAR. In-vivo, the catalytic activity of uPA is this model, a missense mutation in the protease domain of Omi inhibited by its natural inhibitor plasminogen activated inhibitor changes an evolutionary conserved serine to a cysteine (PAI-2) [72,73]. uPA has been largely implicated in metastasis abrogating the serine protease activity without affecting the and the invasion of cancer cells and a relationship with cathepsin ability to interact with IAPs effectively creating an in vivo ‘loss- B has also been suggested. RNA interference-mediated of-function model’ [62]. At the cellular level striated neurons simultaneous down-regulation of uPAR and cathepsin B have exhibit both necrotic and apoptotic features including nuclear been shown to induce caspase-8-mediated apoptosis in human condensation, DNA laddering and early degeneration of glioma cells which is also accompanied by nuclear translocation mitochondria. Mouse Embryonic Fibroblasts (MEFs) from of AIF (Apoptosis Inducing Factor) [74]. mnd2 mice demonstrate increased sensitivity to cell death correlated to increased susceptibility to induce mitochondria 9. Serine proteases and the apoptotic mitochondrion permeability transition (MPT) compared to wild type cells. Given Omi's structural similarity to the bacterial HtrA protease/ A role for serine proteases in early mitochondrial-related chaperone family, it is hypothesised that under normal events has been implied in a number of apoptotic programs, even conditions Omi acts as a stress sensor, preventing the at the level of MOMP induction. For example, a cell death accumulation of misfolded and damaged proteins and preserving program activated in monocytes deprived of IL-3 and in ER mitochondrial membrane integrity and function (Fig. 16). This stressed R6 fibroblasts remains active when caspases are hypothesis has been strengthened by studies in mice entirely inhibited [9]. Co-treatment of the stressed cells with a serine lacking expression of HtrA2/Omi due to the targeted homozy- protease inhibitor prevents apoptosis-associated pyknosis, A.R. O’Connell, C. Stenson-Cox / Biochimica et Biophysica Acta 1773 (2007) 1491–1499 1495 membrane blebbing and phagocytosis when caspase inhibition was detectable and completely abrogated by a serine protease alone does not. Significantly, cytC release is also prevented by inhibitor administrated at the same concentration levels that also the serine protease inhibition implying a mechanism involving prevented the apoptotic cellular and nuclear events. Serine mitochondrial membrane damage. Where exactly serine pro- protease inhibition did not prevent the z-VAD-FMK-dependent teases might function in mitochondrial permeability induction caspase activation or PARP cleavage suggesting that serine was not shown in this study, however evidence from other proteases are activated in parallel but independently to caspases models supports a potential relationship between serine protease [10]. It has been demonstrated that zVAD-FMK can generate activities and Bcl-2 family members (Fig.11). For instance, fluoracetate in cells, which is directly toxic by inhibiting Taxol-induced cell death in human breast carcinoma cells can be aconitase, therefore cell death in the presence of zVAD-FMK prevented by a -like inhibitor that concomitantly may, at times, be due to inhibition of the TCA cycle, rather than blocks Bcl-2 phosphorylation, a post-translational modification activation of other proteases [80,81]. However, this was not the required for its full and potent anti-apoptotic function [75]. Also, case in our study, where cell treatment with a range of z-VAD- a trypsin-like inhibitor prevents cytC release in ethanol-induced FMK concentrations alone did not induce apoptotic or hepatoma cell death, whilst preventing cleavage of Bcl-2 and secondary necrotic features nor was there an additive or Bcl-xl proteins [76]. synergistic rise in apoptotic features observed when adminis- tered prior to staurosporine. 10. Serine proteases and the caspases We have also recently characterised the caspase-dependent activation of the two chymotrypsin-like proteases, p50 and p60 Serine proteases can mediate caspase activation through post- that appear to mediate nuclear events during Jurkat T cell mitochondrial events. For example, apoptosome-stimulated apoptosis (Fig 111) [82]. P60 is constitutively active species caspase-9 processing is preventable by serine protease inhibition whose activity is increased in response to staurosporine whilst [77], Gr B can cleave pro-caspase-3 (Fig. 18) [38] and we and p50 appears active only in the presence of the staurosporine. others have shown that inhibition of chymotrypsin-like Caspase activation is a prequisite for activation of these proteins proteases prevents caspase-3 activation [9,10]. after which they can mediate specific caspase-regulated events, We have also recently characterised three putative serine in particular the late stage morphological features of apoptosis proteases involved in caspase-dependent and independent cell including nuclear fragmentation and condensation, as well as death programmes. P16 is a 16-kDa chymotrypsin-like protein DNA fragmentation. This is further supported by the observa- species that is found basally active in acute myleogenous tion that the serine proteases are activated within the cytosol and leukaemic cells and whose activity is specifically induced in a translocate into the nucleus as apoptosis progresses [82]. time and concentration-dependent manner by the protein Serine proteases may also act upon caspase-independent kinase inhibitor; staurosporine. Detection and characterisation endonucleases, for example, chymotrypsin-like proteases are of p16 was enabled by labelling with a fluorescein-conjugated required for the activation of a manganese-dependent acidic cell permeable compound that covalently binds to the active endonuclease during PBOX-6-induced cell death of Chronic centres of chymotrypsin-like proteases [10]. We hypothesise Myelogenous Leukaemic cells where oligonucleosomal DNA that p16 is a primary mediator of a stauroporine-triggered fragmentation takes place in the absence of caspase and CAD serine protease-mediated cell death pathway that occurs in activation (Fig. 19) [83]. parallel, but independently to caspase-controlled systems in HL-60 cells. The novel pathway accumulates in a loss of cell 11. Serine proteases and receptor-mediated apoptosis volume, nuclear condensation and oligonucleosomal DNA fragmentation (Fig 19) [10]. Serine proteases do not appear to be involved in early events Prior to our study several groups had reported staurosporine- of receptor-mediated death however, cytosolic serine protease triggered caspase-independent progression to cell death in a activity isolated from Fas-treated Jurkat T cells can induce DNA range of cell types including peripheral T lymphocytes, bovine fragmentation implying a role at a later stage [84]. Perhaps more lens epithelial and HL-60 cells, but the identity of the molecules significantly however, is the apparent requirement of serine involved and nature of the non-caspase-mediated events were proteases for optimal signalling in an alternative pathway of unresolved [78,79]. Our initial observation showed that the pan- TNFR1 associated death domain protein (TRADD)-induced caspase inhibitor z-VAD.FMK did not prevent all apoptotic apoptosis [85]. Overexpression of the TRADD core death features activated in staurosporine-treated HL-60 cells, however domain containing a nuclear import sequence results in a it did specifically abrogate staurosporine-induced caspase-3 nuclear localised protein that kills via a caspase-9 and serine activation and cleavage of PARP. To determine the putative non- protease-dependent process, differentiating from the typical caspase protease(s) responsible for the z-VAD-FMK resistant cytosolic caspase-8-dependent route for which caspase activa- apoptotic events, the effects of a panel of cell permeable protease tion appears sufficient. inhibitors were assessed. Results demonstrated that only chymotrypsin-like inhibitors and a pan-serine protease inhibitor 12. Serpins, regulation of serine proteases and cell death could significantly reduce the activated cell shrinkage, nuclear condensation and oligonucleosomal DNA. In addition a rise Regulation of activated proteases is essential in the main- in staurosporine-induced chymotrypsin-like proteolytic activity tenance of normal cellular function and homeostasis. The 1496 A.R. O’Connell, C. Stenson-Cox / Biochimica et Biophysica Acta 1773 (2007) 1491–1499 enzymatic activity of serine proteases is tightly regulated by the , for example human malignant lymphomas specific endogenous serine protease inhibitors, the serpins. express PI-9 [97,98] and murine malignant cells have been Members of the serpin superfamily are known regulators of found to express Spi6 [99]. Other members of the serpin family intracellular proteolysis and have been implicated in regulating such as PAI-2 (plasminogen activator inhibitor 2) and PN-1 the apoptotic process through the inhibition of cytosolic (protease nexin 1) and Spi2A have also been implicated in the proteases [86]. The majority of serpins identified to date behave regulation of cell death [86] and as already mentioned the serine as pseudo-substrates inhibiting activated protease through protease AP24 induces DNA fragmentation indirectly by irreversible complex formation. Proteases bind to a ‘reactive inactivating the serpin leukocyte elastase inhibitor (LEI). centre loop’, inducing a conformational change in the serpin AP24 induces a post-translational modification resulting in a that serves to stabilise the complex rendering it irreversible. molecular weight shift, loss of LEI elastase-inhibiting activity Specificity is dictated primarily through cleavage between two and appearance of L-DNase II activity [64,100]. specific amino acids P1 and P2 within the reactive centre [87]. The viral serpin, cytokine response modifier A (CrmA) was 13. Conclusions and future perspectives originally discovered through its ability to prevent interleukin-1 processing by inhibiting caspase-1 [88,89]. CrmA has since Serine protease involvement in apoptosis is a somewhat been shown to inhibit activated caspase-8 and prevent both controversial topic largely due to the lack of appropriate Fas- and TNF-induced apoptosis [90–92]. Significantly, CrmA biological tools with which to study them and the relatively is also an inhibitor of Gr B displaying an Asp at the P1 small number of pro-apoptotic serine proteases identified to position in the reactive center loop [88,93]. CrmA can abro- date. However a preponderance of evidence over the last decade gate Gr B-mediated apoptosis and expression of CrmA renders continues to imply their involvement in cell death where they target cells resistant to Gr B-induced cell death by CLs [90]. The demonstrate a range of molecular mechanisms, specificity and cytosolic serpin protease inhibitor 9 (PI-9) is the only known regulation. It is to be expected that advances in biochemistry human protein that can specifically inhibit granzyme B activity and proteomics can be harnessed to extract and characterise and exhibits a greater potency than CrmA [94]. Although it novel pro-apoptotic serine proteases that are responsible for shares many similarities to the viral-encoded CrmA, PI-9 supporting and propagating caspase and non-caspase-mediated contains a Glu rather than Asp at the crucial P1 position and cell death programs. this appears to directly affect its specificity. Cells expressing PI- 9 are protected against Gr B-induced apoptosis, however PI-9 Acknowledgements does not confer resistance to Fas-induced apoptosis. The Glu at P1 confers specificity for Gr B only and the serpin is a poor The authors would like to acknowledge funding by SFI, substrate for caspases [95]. It has been postulated that the IRCSET, HEA and Enterprise Ireland. The authors also wish to inhibition of Gr B-mediated, but not Fas-induced apoptosis by thank Dr. Ralf Zwacka and Dr. Andrea Mohr for critical reading PI-9 may serve to protect CLs from apoptosis following of the manuscript. cytotoxic cell degranulation whilst allowing the deletion of cells from the immune system via the Fas pathway [95]. References Furthermore, the in vivo distribution of PI-9 mRNA demon- strates an expression pattern restricted largely to the immune [1] J.F. Kerr, A.H. Wyllie, A.R. Currie, Apoptosis: a basic biological tissue, with expression primarily in the lymphocytes. The phenomenon with wide-ranging implications in tissue kinetics, Br. J. – restricted expression profile may explain why cytotoxic cells are Cancer 26 (1972) 239 257. [2] N.N. Danial, S.J. Korsmeyer, Cell death: critical control points, Cell 116 not damaged by their own Gr B during elimination of target cells, (2004) 205–219. with CL synthesising their own serpins that bind and neutralize [3] P. 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