Bacterial secreted effectors and caspase‐3 interactions

Apoptosis is a critical process that intrinsically links organism survival to its ability to induce controlled death. Thus, functional apoptosis allows organisms to remove perceived threats to their survival by targeting those cells that it determines pose a direct risk. Central to this process are apoptotic caspases, enzymes that form a signalling cascade, converting danger signals via initiator caspases into activation of the executioner caspase, caspase‐3. This enzyme begins disassembly of the cell by activating DNA degrading enzymes and degrading the cellular architecture. Interaction of pathogenic bacteria with caspases, and in particular, caspase‐3, can therefore impact both host cell and bacterial survival. With roles outside cell death such as cell differentiation, control of signalling pathways and immunomodulation also being described for caspase‐3, bacterial interactions with caspase‐3 may be of far more significance in infection than previously recognized. In this review, we highlight the ways in which bacterial pathogens have evolved to subvert caspase‐3 both through effector proteins that directly interact with the enzyme or by modulating pathways that influence its activation and activity.     

Quantitative analysis of pathways controlling extrinsic apoptosis in single cells

Apoptosis in response to TRAIL or TNF requires the activation of initiator caspases, which then activate the effector caspases that dismantle cells and cause death. However, little is known about the dynamics and regulatory logic linking initiators and effectors. Using a combination of live-cell reporters, flow cytometry, and immunoblotting, we find that initiator caspases are active during the long and variable delay that precedes mitochondrial outer membrane permeabilization (MOMP) and effector caspase activation. When combined with a mathematical model of core apoptosis pathways, experimental perturbation of regulatory links between initiator and effector caspases reveals that XIAP and proteasome-dependent degradation of effector caspases are important in restraining activity during the pre-MOMP delay. We identify conditions in which restraint is impaired, creating a physiologically indeterminate state of partial cell death with the potential to generate genomic instability. Together, these findings provide a quantitative picture of caspase regulatory networks and their failure modes.     

Fas death receptor signalling: roles of Bid and XIAP

Fas (also called CD95 or APO-1), a member of a subgroup of the tumour necrosis factor receptor superfamily that contain an intracellular death domain, can initiate apoptosis signalling and has a critical role in the regulation of the immune system. Fas-induced apoptosis requires recruitment and activation of the initiator caspase, caspase-8 (in humans also caspase-10), within the death-inducing signalling complex. In so-called type 1 cells, proteolytic activation of effector caspases (-3 and -7) by caspase-8 suffices for efficient apoptosis induction. In so-called type 2 cells, however, killing requires amplification of the caspase cascade. This can be achieved through caspase-8-mediated proteolytic activation of the pro-apoptotic Bcl-2 homology domain (BH)3-only protein BH3-interacting domain death agonist (Bid), which then causes mitochondrial outer membrane permeabilisation. This in turn leads to mitochondrial release of apoptogenic proteins, such as cytochrome c and, pertinent for Fas death receptor (DR)-induced apoptosis, Smac/DIABLO (second mitochondria-derived activator of caspase/direct IAP binding protein with low Pi), an antagonist of X-linked inhibitor of apoptosis (XIAP), which imposes a brake on effector caspases. In this review, written in honour of Juerg Tschopp who contributed so much to research on cell death and immunology, we discuss the functions of Bid and XIAP in the control of Fas DR-induced apoptosis signalling, and we speculate on how this knowledge could be exploited to develop novel regimes for treatment of cancer.     

A dimeric Smac/diablo peptide directly relieves caspase-3 inhibition by XIAP. Dynamic and cooperative regulation of XIAP by Smac/Diablo

Caspase activation, the executing event of apoptosis, is under deliberate regulation. IAP proteins inhibit caspase activity, whereas Smac/Diablo antagonizes IAP. XIAP, a ubiquitous IAP, can inhibit both caspase-9, the initiator caspase of the mitochondrial apoptotic pathway, and the downstream effector caspases, caspase-3 and caspase-7. Smac neutralizes XIAP inhibition of caspase-9 by competing for binding of the BIR3 domain of XIAP with caspase-9, whereas how Smac liberates effector caspases from XIAP inhibition is not clear. It is generally believed that binding of Smac with IAP generates a steric hindrance that prevents XIAP from inhibiting effector caspases, and therefore small molecule mimics of Smac are not able to reverse inhibition of the effector caspases. Surprisingly, we show here that binding of a dimeric Smac N-terminal peptide with the BIR2 domain of XIAP effectively antagonizes inhibition of caspase-3 by XIAP. Further, we defined the dynamic and cooperative interaction of Smac with XIAP: binding of Smac with the BIR3 domain anchors the subsequent binding of Smac with the BIR2 domain, which in turn attenuates the caspase-3 inhibitory function of XIAP. We also show that XIAP homotrimerizes via its C-terminal Ring domain, making its inhibitory activity toward caspase-3 more susceptible to Smac.     

Cellular, biochemical, and genetic analysis of mechanism of small molecule IAP inhibitors

XIAP is member of the IAP family of anti-apoptotic proteins and is known for its ability to bind and suppress caspase family cell death proteases. A phenylurea series of chemical inhibitors of XIAP was recently generated by our laboratories (Schimmer, A. D., Welsh, K., Pinilla, C., Bonneau, M., Wang, Z., Pedersen, I. M., Scott, F. L., Glinsky, G. V., Scudiero, D. A., Sausville, E., Salvesen, G., Nefzi, A., Ostresh, J. M., Houghten, R. A., and Reed, J. C. (2004) Cancer Cell 5, 25-35). We examined the mechanisms of action of these chemical compounds using biochemical, molecular biological, and genetic methods. Active phenylurea-based compounds dissociated effector protease caspase-3 but not initiator protease caspase-9 from XIAP in vitro and restored caspase-3 but not caspase-9 enzymatic activity. When applied to tumor cell lines in culture, active phenylurea-based compounds induced apoptosis in a rapid, concentration-dependent manner, associated with activation of cellular caspases. Apoptosis induced by active phenylurea-based compounds was blocked by chemical inhibitors of caspases, with inhibitors of downstream effector caspases displaying more effective suppression than inhibitors of upstream initiator caspases. Phenylurea-based XIAP antagonists induced apoptosis (defined by annexin V staining) prior to mitochondrial membrane depolarization, in contrast to cytotoxic anticancer drugs. Consistent with these findings, apoptosis induced by phenylurea-based compounds was not altered by genetic alterations in the expression of Bcl-2 family proteins that control mitochondria-dependent cell death pathways, including over-expression of anti-apoptotic proteins Bcl-2 or Bcl-X(L) and genetic ablation of pro-apoptotic proteins Bax and Bak. Conversely, conditional over-expression of an active fragment of XIAP or genetic ablation of XIAP expression altered the apoptosis dose-response of the compounds. Altogether, these findings indicate that phenylurea-based XIAP antagonists block interaction of downstream effector caspases with XIAP, thus inducing apoptosis of tumor cell lines through a caspase-dependent, Bcl-2/Bax-independent mechanism.     

Overexpression of Par‐4 sensitizes TRAIL‐induced apoptosis via inactivation of NF‐κB and Akt signaling pathways in renal cancer cells

The prostate‐apoptosis‐response‐gene‐4 (Par‐4) is up‐regulated in prostate cells undergoing programmed cell death. Furthermore, Par‐4 protein has been shown to function as an effector of cell death in response to various apoptotic stimuli that trigger mitochondria and membrane receptor‐mediated cell death pathways. In this study, we investigated how Par‐4 modulates TRAIL‐mediated apoptosis in TRAIL‐resistant Caki cells. Par‐4 overexpressing cells were strikingly sensitive to apoptosis induced by TRAIL compared with control cells. Par‐4 overexpressing Caki cells treated with TRAIL showed an increased activation of the initiator caspase‐8 and the effector caspase‐3, together with an enforced cleavage of XIAP and c‐FLIP. TRAIL‐induced reduction of XIAP and c‐FLIP protein levels in Par‐4 overexpressing cells was prevented by z‐VAD pretreatment. In addition, the surface DR5 protein level was increased in TRAIL‐treated Par‐4 overexpressing cells. Interestingly, even though a deletion of leucine zipper domain in Par‐4 recovered Bcl‐2 level to basal level induced by wild type Par‐4, it partly decreased sensitivity to TRAIL in Caki cells. In addition, exposure of Caki/Par‐4 cells to TRAIL led to reduction of phosphorylated Akt levels, but deletion of leucine zipper domain of Par‐4 did not affect these phosphorylated Akt levels. In conclusion, we here provide evidence that ectopic expression of Par‐4 sensitizes Caki cells to TRAIL via modulation of multiple targets, including DR5, Bcl‐2, Akt, and NF‐κB. J. Cell. Biochem. 109: 885–895, 2010. © 2010 Wiley‐Liss, Inc.     

Interdimer processing and linearity of procaspase-3 activation. A unifying mechanism for the activation of initiator and effector caspases

Caspase activation during apoptosis occurs in a cascade from the initiator caspase(s) (e.g. caspase-8) to the effector caspases (e.g. caspase-3), which ensures the generation of large amounts of active caspases to dismantle cells. However, the mechanism that safeguards against inadvertent caspase activation is not well understood. Previous studies have suggested that the activation of procaspase-8 is mediated by cross-cleavage of precursor dimers, formed upon apoptosis induction, which are not only enzymatically competent but also highly susceptible to cleavage, and that procaspase-8 activation is a linear process without self-amplification. Effector procaspases constitutively exist as dimers and their activation is started by trans-cleavage by an initiator caspase followed by autocleavage of effector caspases. Here we show that the dimerization of caspase-3 molecules through their protease domains is required for their processing by initiator caspases. The subsequent autoprocessing takes place through cleavage between the dimeric intermediates. Moreover, mature caspase-3 fails to process its own precursor. Thus, despite a marked difference in the generation of active intermediates, the activation of initiator and effector caspases shares the features of interdimer cleavage and lack of self-amplification. These features may be important in preventing accidental cell death.     

IAP-antagonists exhibit non-redundant modes of action through differential DIAP1 binding

The Drosophila inhibitor of apoptosis protein DIAP1 ensures cell viability by directly inhibiting caspases. In cells destined to die this IAP-mediated inhibition of caspases is overcome by IAP-antagonists. Genetic evidence indicates that IAP-antagonists are non-equivalent and function synergistically to promote apoptosis. Here we provide biochemical evidence for the non-equivalent mode of action of Reaper, Grim, Hid and Jafrac2. We find that these IAP-antagonists display differential and selective binding to specific DIAP1 BIR domains. Consistently, we show that each DIAP1 BIR region associates with distinct caspases. The differential DIAP1 BIR interaction seen both between initiator and effector caspases and within IAP-antagonist family members suggests that different IAP-antagonists inhibit distinct caspases from interacting with DIAP1. Surprisingly, we also find that the caspase-binding residues of XIAP predicted to be strictly conserved in caspase-binding IAPs, are absent in DIAP1. In contrast to XIAP, residues C-terminal to the DIAP1 BIR1 domain are indispensable for caspase association. Our studies on DIAP1 and caspases expose significant differences between DIAP1 and XIAP suggesting that DIAP1 and XIAP inhibit caspases in different ways.     

Molecular determinants of the caspase-promoting activity of Smac/DIABLO and its role in the death receptor pathway

Smac/DIABLO is a mitochondrial protein that is released along with cytochrome c during apoptosis and promotes cytochrome c-dependent caspase activation by neutralizing inhibitor of apoptosis proteins (IAPs). We provide evidence that Smac/DIABLO functions at the levels of both the Apaf-1-caspase-9 apoptosome and effector caspases. The N terminus of Smac/DIABLO is absolutely required for its ability to interact with the baculovirus IAP repeat (BIR3) of XIAP and to promote cytochrome c-dependent caspase activation. However, it is less critical for its ability to interact with BIR1/BIR2 of XIAP and to promote the activity of the effector caspases. Consistent with the ability of Smac/DIABLO to function at the level of the effector caspases, expression of a cytosolic Smac/DIABLO in Type II cells allowed TRAIL to bypass Bcl-xL inhibition of death receptor-induced apoptosis. Combined, these data suggest that Smac/DIABLO plays a critical role in neutralizing IAP inhibition of the effector caspases in the death receptor pathway of Type II cells.     

Mechanisms mediating caspase activation in cell death

The initial activation of a caspase in a caspase cascade is a crucial event that determines whether a cell will ultimately undergo cell death. Although each cell contains a number of different caspases, only a small subset may be required for apoptosis in response to a specific stimulus. It now seems that each caspase cascade has two types of caspases involved, the upstream or class I caspases, and the downstream or class II caspases. Class I caspases are characterised by long amino-terminal prodomains that carry specific protein - protein interaction domains which mediate oligomerisation of caspases, often assisted by specific adaptor molecules. Oligomerisation appears to be sufficient for autocatalytic activation of class I caspases. Once the first caspase in the pathway has been activated, it processes downstream caspases initiating a cascade of amplifying events that lead to the apoptotic death of a cell. This article reviews our current understanding of mechanisms that mediate the activation of caspases.     

Caspase inhibitor P35 and inhibitor of apoptosis Op-IAP block in vivo proteolytic activation of an effector caspase at different steps

Signal-induced activation of caspases, the critical protease effectors of apoptosis, requires proteolytic processing of their inactive proenzymes. Consequently, regulation of procaspase processing is critical to apoptotic execution. We report here that baculovirus pancaspase inhibitor P35 and inhibitor of apoptosis Op-IAP prevent caspase activation in vivo, but at different steps. By monitoring proteolytic processing of endogenous Sf-caspase-1, an insect group II effector caspase, we show that Op-IAP blocked the first activation cleavage at TETD downward arrowG between the large and small caspase subunits. In contrast, P35 failed to affect this cleavage, but functioned downstream to block maturation cleavages (DXXD downward arrow(G/A)) of the large subunit. Substitution of P35's reactive site residues with TETDG failed to increase its effectiveness for blocking TETD downward arrowG processing of pro-Sf-caspase-1, despite wild-type function for suppressing apoptosis. These data are consistent with the involvement of a novel initiator caspase that is resistant to P35, but directly or indirectly inhibitable by Op-IAP. The conservation of TETD downward arrowG processing sites among insect effector caspases, including Drosophila drICE and DCP-1, suggests that in vivo activation of these group II caspases involves a P35-insensitive caspase and supports a model wherein apical and effector caspases function through a proteolytic cascade to execute apoptosis in insects.     

Modeling a Snap-Action, Variable-Delay Switch Controlling Extrinsic Cell Death

When exposed to tumor necrosis factor (TNF) or TNF-related apoptosis-inducing ligand (TRAIL), a closely related death ligand and investigational therapeutic, cells enter a protracted period of variable duration in which only upstream initiator caspases are active. A subsequent and sudden transition marks activation of the downstream effector caspases that rapidly dismantle the cell. Thus, extrinsic apoptosis is controlled by an unusual variable-delay, snap-action switch that enforces an unambiguous choice between life and death. To understand how the extrinsic apoptosis switch functions in quantitative terms, we constructed a mathematical model based on a mass-action representation of known reaction pathways. The model was trained against experimental data obtained by live-cell imaging, flow cytometry, and immunoblotting of cells perturbed by protein depletion and overexpression. The trained model accurately reproduces the behavior of normal and perturbed cells exposed to TRAIL, making it possible to study switching mechanisms in detail. Model analysis shows, and experiments confirm, that the duration of the delay prior to effector caspase activation is determined by initiator caspase-8 activity and the rates of other reactions lying immediately downstream of the TRAIL receptor. Sudden activation of effector caspases is achieved downstream by reactions involved in permeabilization of the mitochondrial membrane and relocalization of proteins such as Smac. We find that the pattern of interactions among Bcl-2 family members, the partitioning of Smac from its binding partner XIAP, and the mechanics of pore assembly are all critical for snap-action control.     

N‐(3‐Oxo‐acyl)‐homoserine lactone induces apoptosis primarily through a mitochondrial pathway in fibroblasts

N‐(3‐Oxododecanoyl)‐l ‐homoserine lactone (C12) is produced by Pseudomonas aeruginosa to function as a quorum‐sensing molecule for bacteria–bacteria communication. C12 is also known to influence many aspects of human host cell physiology, including induction of cell death. However, the signalling pathway(s) leading to C12‐triggered cell death is (are) still not completely known. To clarify cell death signalling induced by C12, we examined mouse embryonic fibroblasts deficient in “initiator” caspases or “effector” caspases. Our data indicate that C12 selectively induces the mitochondria‐dependent intrinsic apoptotic pathway by quickly triggering mitochondrial outer membrane permeabilisation. Importantly, the activities of C12 to permeabilise mitochondria are independent of activation of both “initiator” and “effector” caspases. Furthermore, C12 directly induces mitochondrial outer membrane permeabilisation in vitro. Overall, our study suggests a mitochondrial apoptotic signalling pathway triggered by C12, in which C12 or its metabolite(s) acts on mitochondria to permeabilise mitochondria, leading to activation of apoptosis.     

Caspase-8 and Apaf-1-independent caspase-9 activation in Sendai virus-infected cells

Apoptotic cell death is of central importance in the pathogenesis of viral infections. Activation of a cascade of cysteine proteases, i.e. caspases, plays a key role in the effector phase of virus-induced apoptosis. However, little is known about pathways leading to the activation of initiator caspases in virus-infected host cells. Recently, we have shown that Sendai virus (SeV) infection triggers apoptotic cell death by activation of the effector caspase-3 and initiator caspase-8. We now investigated mechanisms leading to the activation of another initiator caspase, caspase-9. Unexpectedly we found that caspase-9 cleavage is not dependent on the presence of active caspases-3 or -8. Furthermore, the presence of caspase-9 in mouse embryonic fibroblast (MEF) cells was a prerequisite for Sendai virus-induced apoptotic cell death. Caspase-9 activation occurred without the release of cytochrome c from mitochondria and was not dependent on the presence of Apaf-1 or reactive oxygen intermediates. Our results therefore suggest an alternative mechanism for caspase-9 activation in virally infected cells beside the well characterized pathways via death receptors or mitochondrial cytochrome c release.     

ROS-triggered caspase 2 activation and feedback amplification loop in beta-carotene-induced apoptosis

Reactive oxygen species (ROS) and caspases 8, 9, and 3 are reported to be crucial players in apoptosis induced by various stimuli. Recently, caspase 2 has been implicated in stress-induced apoptosis but the exact mechanism remains unclear. In this study, we report that ROS generation led to activation of caspase 2 during beta-carotene-induced apoptosis in the human leukemic T cell line Molt 4. The apoptosis progressed by simultaneous activation of caspases 8 and 9, and a cross talk between these initiator caspases was mediated by the proapoptotic protein Bid. Inhibition of caspases 2, 8, 9, and 3 independently suppressed the caspase cascade. The kinetics and function of caspase 2 were similar to those of caspase 3, suggesting its role as an effector caspase. Interestingly, beta-carotene-induced apoptosis was caspase 2 dependent but caspase 3 independent. The study also revealed cleavage of the antiapoptotic protein BclXL as an important event during apoptosis, which was regulated by ROS. The mechanistic studies identify a functional link between ROS and the caspase cascade involving caspase 2 and cleavage of BclXL. The interdependence of caspases 8, 9, 2, and 3 in the cascade provides evidence for the presence of an extensive feedback amplification loop in beta-carotene-induced apoptosis in Molt 4 cells.     

Simultaneous imaging of initiator/effector caspase activity and mitochondrial membrane potential during cell death in living HeLa cells

A family of cystein proteases, the caspases, plays a central role in mediating cell death. In this study, we measured the activation of the initiator and effector caspase in real time, and studied the relationship between caspase activity and mitochondrial membrane potential in living cells by means of bioimaging. We also designed and developed a fluorescence resonance energy transfer (FRET)-based genetically encoded fluorescent indicator, which consisted of yellow fluorescent protein (YFP), a peptide sequence which can be cleaved by specific caspases, and cyan fluorescent protein (CFP). Two peptide sequences which could be cleaved by initiator caspases and effector caspases, respectively, were used. Simultaneous real-time measurements of the caspase activity and mitochondrial membrane potential in the cells treated with TNF-alpha and staurosporine revealed that dying cells showed caspase activation and mitochondrial depolarization, and that these events, however, were not firmly linked. Although it takes anywhere from 1 to over 10 h after the addition of the cell death inducer for the caspases to begin to be activated, initiator caspases and effector caspases are activated within a short period of time at the last stage in the entire process leading to cell death.     

Caspase activation: revisiting the induced proximity model

Caspases execute cell death. The mechanism of effector caspase activation primarily involves reorganization of active site loops following the activation cleavage. The Induced Proximity hypothesis, originally proposed to explain the activation of initiator caspases, has recently been reinterpreted to be proximity-driven dimerization of initiator caspases, and consequently their activation. The evidence supporting these models is critically evaluated and other possible mechanisms for initiator caspase activation are discussed.     

Synergetic Effects of Caspase 3 and μ-Calpain in XIAP-Breakdown upon Focal Cerebral Ischemia

Dysregulation of apoptosis is involved in a wide spectrum of disease ranging from proliferative to neurodegenerative disorders. The recently discovered X-linked inhibitor of apoptosis protein (XIAP) is among the most potent inhibitors of apoptosis. This protein binds to and inhibits both initiator caspases and effector caspases such as caspase-3. The aim of this study was to investigate the relationships between XIAP-breakdown, caspase activation in the development of delayed infarct upon ischemia. We demonstrated that endogenous XIAP is cleaved at least into two fragments during reperfusion following the ischemic insult. The two fragments produced seem to be related to caspase-3 and μ-calpain activities, which are massively enhanced in tissues challenged by ischemia. Therefore, degradation of XIAP by μ-calpain in our system may decrease the activation threshold of caspase-3 normally held in check by the IAPs and/or lead to auto-activation of other caspases. Special issue in honor of Naren Banik.     

Caspase signalling in the absence of apoptosis drives Jnk‐dependent invasion

Tumours evolve several mechanisms to evade apoptosis, yet many resected carcinomas show significantly elevated caspase activity. Moreover, caspase activity is positively correlated with tumour aggression and adverse patient outcome. These observations indicate that caspases might have a functional role in promoting tumour invasion and metastasis. Using a Drosophila model of invasion, we show that precise effector caspase activity drives cell invasion without initiating apoptosis. Affected cells express the matrix metalloprotinase Mmp1 and invade by activating Jnk. Our results link Jnk and effector caspase signalling during the invasive process and suggest that tumours under apoptotic stresses from treatment, immune surveillance or intrinsic signals might be induced further along the metastatic cascade.     

Requirement of both the second and third BIR domains for the relief of X-linked inhibitor of apoptosis protein (XIAP)-mediated caspase inhibition by Smac

The inhibitor of apoptosis proteins (IAP) are endogenous caspase inhibitors in the metazoan and characterized by the presence of baculoviral IAP repeats (BIR). X-linked IAP (XIAP) contains three BIR domains and directly inhibits effector caspases such as caspase-7 via a linker_BIR2 fragment and initiator caspases such as caspase-9 via the BIR3 domain. A mitochondrial protein Smac/DIABLO, which is released during apoptosis, antagonizes XIAP-mediated caspase inhibition by interacting directly with XIAP. Here, using glutathione S-transferase pulldown and caspase activity assay, we show that Smac is ineffective in relieving either caspase-7 or caspase-9 inhibition by XIAP domain fragments. In addition, Smac forms a ternary complex with caspase-7 and linker_BIR2, suggesting that Smac/linker_BIR2 interaction does not sterically exclude linker_BIR2/caspase-7 interaction. However, Smac is effective in removing caspase-7 and caspase-9 inhibition by XIAP fragments containing both the BIR2 and BIR3 domains. Surface plasmon resonance measurements show that Smac interacts with the BIR2 or BIR3 domain in micromolar dissociation constants. On the other hand, Smac interacts with an XIAP construct containing both BIR2 and BIR3 domains in a subnanomolar dissociation constant by the simultaneous interaction of the Smac dimer with the BIR2 and BIR3 domains of a single XIAP molecule. This 2:1 Smac/XIAP interaction not only possesses enhanced affinity but also sterically excludes XIAP/caspase-7 interaction, demonstrating the requirement of both BIR2 and BIR3 domains for Smac to relieve XIAP-mediated caspase inhibition.