Greasing the path to BAX/BAK activation.

BAX/BAK activation leading to mitochondrial outer-membrane permeabilization is a key commitment point in apoptosis. Chipuk et al. now identify two sphingolipids as specific cofactors for BAX/BAK activation that lower the threshold for apoptosis-associated cytochrome c release. Association of mitochondria with other cellular membrane compartments is required for BAK/BAX exposure to these sphingolipids.

Unleashing dendritic cell-mediated tumor clearance by targeting Bcl-2.

Bcl-2 family proteins serve as key regulators of apoptosis and are frequently overexpressed in cancer. Consequently, small-molecule Bcl-2-antagonists (BH3 mimetics) have emerged as a new class of targeted therapeutics. A recent study by Zhao et al. has unexpectedly found that BH3 mimetics can also activate dendritic cells (DCs) to prime for T cell-mediated tumor clearance.

An Inflammatory Perspective on Necroptosis.

Necroptosis (programmed necrosis) occurs in response to TNF, Fas, or TRAIL, as well as certain TLR ligands, when caspase activity required for apoptosis is blocked. Necroptosis is typically considered a highly pro-inflammatory mode of cell death, due to release of intracellular "danger signals" that promote inflammation. However, because most pro-necroptotic stimuli are intrinsically highly pro-inflammatory-due to their ability to initiate the synthesis of numerous cytokines and chemokines-the inflammatory consequences of necroptosis are complex. Here, we suggest that necroptosis might have anti-inflammatory effects in certain settings, through curbing excessive TNF- or TLR-induced inflammatory cytokine production.

Caspase-8 Acts in a Non-enzymatic Role as a Scaffold for Assembly of a Pro-inflammatory "FADDosome" Complex upon TRAIL Stimulation.

TRAIL is a potent inducer of apoptosis and has been studied almost exclusively in this context. However, TRAIL can also induce NFκB-dependent expression of multiple pro-inflammatory cytokines and chemokines. Surprisingly, whereas inhibition of caspase activity blocked TRAIL-induced apoptosis, but not cytokine production, knock down or deletion of caspase-8 suppressed both outcomes, suggesting that caspase-8 participates in TRAIL-induced inflammatory signaling in a scaffold role. Consistent with this, introduction of a catalytically inactive caspase-8 mutant into CASP-8 null cells restored TRAIL-induced cytokine production, but not cell death. Furthermore, affinity precipitation of the native TRAIL receptor complex revealed that pro-caspase-8 was required for recruitment of RIPK1, via FADD, to promote NFκB activation and pro-inflammatory cytokine production downstream. Thus, caspase-8 can serve in two distinct roles in response to TRAIL receptor engagement, as a scaffold for assembly of a Caspase-8-FADD-RIPK1 "FADDosome" complex, leading to NFκB-dependent inflammation, or as a protease that promotes apoptosis.

Proteolytic Processing of Interleukin-1 Family Cytokines: Variations on a Common Theme.

Members of the extended interleukin-1 (IL-1) cytokine family, such as IL-1, IL-18, IL-33, and IL-36, play a pivotal role in the initiation and amplification of immune responses. However, deregulated production and/or activation of these cytokines can lead to the development of multiple inflammatory disorders. IL-1 family members share a broadly similar domain organization and receptor signaling pathways. Another striking similarity between IL-1 family members is the requirement for proteolytic processing in order to unlock their full biological potential. Although much emphasis has been put on the role of caspase-1, another emerging theme is the involvement of neutrophil- and mast cell-derived proteases in IL-1 family cytokine processing. Elucidating the regulation of IL-1 family members by proteolytic processing is of great interest for understanding inflammation and immunity. Here, we review the identity of the proteases involved in the proteolytic processing of IL-1 family cytokines and the therapeutic implications in inflammatory disease.

A chromatin-independent role of Polycomb-like 1 to stabilize p53 and promote cellular quiescence.

Polycomb-like proteins 1-3 (PCL1-3) are substoichiometric components of the Polycomb-repressive complex 2 (PRC2) that are essential for association of the complex with chromatin. However, it remains unclear why three proteins with such apparent functional redundancy exist in mammals. Here we characterize their divergent roles in both positively and negatively regulating cellular proliferation. We show that while PCL2 and PCL3 are E2F-regulated genes expressed in proliferating cells, PCL1 is a p53 target gene predominantly expressed in quiescent cells. Ectopic expression of any PCL protein recruits PRC2 to repress the INK4A gene; however, only PCL2 and PCL3 confer an INK4A-dependent proliferative advantage. Remarkably, PCL1 has evolved a PRC2- and chromatin-independent function to negatively regulate proliferation. We show that PCL1 binds to and stabilizes p53 to induce cellular quiescence. Moreover, depletion of PCL1 phenocopies the defects in maintaining cellular quiescence associated with p53 loss. This newly evolved function is achieved by the binding of the PCL1 N-terminal PHD domain to the C-terminal domain of p53 through two unique serine residues, which were acquired during recent vertebrate evolution. This study illustrates the functional bifurcation of PCL proteins, which act in both a chromatin-dependent and a chromatin-independent manner to regulate the INK4A and p53 pathways.

IL-1 family cytokines serve as 'activity recognition receptors' for aberrant protease activity indicative of danger.

Members of the extended IL-1 cytokine family play key roles as instigators of inflammation in numerous infectious and sterile injury contexts and are highly enriched at barrier surfaces such as the skin, lungs and intestinal mucosa. Because IL-1 family cytokines do not possess conventional ER-golgi trafficking and secretory signals, these cytokines are typically released into the extracellular space due to tissue damage resulting in necrosis, or pathogen detection resulting in pyroptosis. The latter feature, in combination with other factors, suggests that IL-1 family cytokines serve as canonical damage-associated molecular patterns (DAMPs), which instigate inflammation in response to tissue damage. However, IL-1 family cytokines also require a proteolytic activation step and diverse intracellular, extracellular and non-self proteases have been identified that are capable of processing and activating members of this family. This suggests that IL-1 family members function as sentinels for aberrant protease activity, which is frequently associated with infection or tissue damage. Here, we overview the diversity of proteases implicated in the activation of IL-1 family cytokines and suggest that this ancient cytokine family may have evolved to complement 'pattern recognition receptors', by serving as 'activity recognition receptors' enabling the detection of aberrant enzyme activity indicative of 'danger'.

Bcl-2 family proteins participate in mitochondrial quality control by regulating Parkin/PINK1-dependent mitophagy.

Mitophagy facilitates the selective elimination of impaired or depolarized mitochondria through targeting the latter to autophagosomes. Parkin becomes localized to depolarized mitochondria in a PINK1-dependent manner and polyubiquitinates multiple mitochondrial outer membrane proteins. This permits ubiquitin-binding proteins (e.g., p62 and NBR1) to target impaired mitochondria to autophagosomes via Atg8/LC3II. Bcl-2 family proteins regulate mitochondrial outer membrane permeabilization during apoptosis and can also influence macroautophagy via interactions with Beclin-1. Here, we show that Parkin-dependent mitophagy is antagonized by prosurvival members of the Bcl-2 family (e.g., Bcl-xL and Mcl-1) in a Beclin-1-independent manner. Bcl-2 proteins suppressed mitophagy through inhibition of Parkin translocation to depolarized mitochondria. Consistent with this, Parkin translocation to mitochondria was enhanced by BH3-only proteins or a BH3-only mimetic. Taken together with their role as regulators of apoptosis-associated mitochondrial permeabilization, as well as mitochondrial fission/fusion dynamics, this suggests that Bcl-2 family proteins act as global regulators of mitochondrial homeostasis.

Fas/CD95-induced chemokines can serve as "find-me" signals for apoptotic cells.

Apoptosis is commonly thought to represent an immunologically silent or even anti-inflammatory mode of cell death, resulting in cell clearance in the absence of explicit activation of the immune system. However, here we show that Fas/CD95-induced apoptosis is associated with the production of an array of cytokines and chemokines, including IL-6, IL-8, CXCL1, MCP-1, and GMCSF. Fas-induced production of MCP-1 and IL-8 promoted chemotaxis of phagocytes toward apoptotic cells, suggesting that these factors serve as "find-me" signals in this context. We also show that RIPK1 and IAPs are required for optimal production of cytokines and chemokines in response to Fas receptor stimulation. Consequently, a synthetic IAP antagonist potently suppressed Fas-dependent expression of multiple proinflammatory mediators and inhibited Fas-induced chemotaxis. Thus, in addition to provoking apoptosis, Fas receptor stimulation can trigger the secretion of chemotactic factors and other immunologically active proteins that can influence immune responsiveness toward dying cells.

Apoptosis: controlled demolition at the cellular level.

Apoptosis is characterized by a series of dramatic perturbations to the cellular architecture that contribute not only to cell death, but also prepare cells for removal by phagocytes and prevent unwanted immune responses. Much of what happens during the demolition phase of apoptosis is orchestrated by members of the caspase family of cysteine proteases. These proteases target several hundred proteins for restricted proteolysis in a controlled manner that minimizes damage and disruption to neighbouring cells and avoids the release of immunostimulatory molecules.

A perspective on mammalian caspases as positive and negative regulators of inflammation.

Members of the caspase family of cysteine proteases coordinate the morphological and biochemical events that typify apoptosis. However, neutralization of caspase activity in mammals fails to block death in response to most proapoptotic stimuli. This is because many cell death triggers provoke mitochondrial dysfunction upstream of caspase activation as a consequence of BAX/BAK channel opening. Although genetic or pharmacological inactivation of caspases fails to block cell death in most instances, it does convert the phenotype from apoptosis to necrosis. This has important implications for how the immune system responds to such cells, as necrotic cells provoke inflammation whereas apoptotic cells typically do not. Here, we propose an alternative perspective on apoptosis-associated caspase function by suggesting that these proteases are activated, not to kill, but to extinguish the proinflammatory properties of dying cells. This perspective unifies the mammalian caspase family as either positive or negative regulators of inflammation.

Autophagy in malignant transformation and cancer progression.

Autophagy plays a key role in the maintenance of cellular homeostasis. In healthy cells, such a homeostatic activity constitutes a robust barrier against malignant transformation. Accordingly, many oncoproteins inhibit, and several oncosuppressor proteins promote, autophagy. Moreover, autophagy is required for optimal anticancer immunosurveillance. In neoplastic cells, however, autophagic responses constitute a means to cope with intracellular and environmental stress, thus favoring tumor progression. This implies that at least in some cases, oncogenesis proceeds along with a temporary inhibition of autophagy or a gain of molecular functions that antagonize its oncosuppressive activity. Here, we discuss the differential impact of autophagy on distinct phases of tumorigenesis and the implications of this concept for the use of autophagy modulators in cancer therapy.

Oncogenic Ras-induced expression of Noxa and Beclin-1 promotes autophagic cell death and limits clonogenic survival.

Deregulated oncogenes such as MYC and RAS are typically insufficient to transform cells on their own due to the activation of pathways that restrain proliferation. Previous studies have shown that oncogenic H-Ras can induce proliferative arrest or senescence, depending on the cellular context. Here, we show that deregulated H-Ras activity can also lead to caspase-independent cell death with features of autophagy. Ras-induced autophagy was associated with upregulation of the BH3-only protein Noxa as well as the autophagy regulator Beclin-1. Silencing of Noxa or Beclin-1 expression reduced Ras-induced autophagy and increased clonogenic survival. Ras-induced cell death was also inhibited by coexpression of Bcl-2 family members that inhibit Beclin-1 function. Ras-induced autophagy was associated with Noxa-mediated displacement of the Bcl-2 family member, Mcl-1, from Beclin-1. Thus, Ras-induced expression of Noxa and Beclin-1 promotes autophagic cell death, which represents a mechanism to limit the oncogenic potential of deregulated Ras signals.

Staying alive: defensive strategies in the BCL-2 family playbook.

Much debate surrounds how prosurvival members of the BCL-2 family repress opening of the BAX/BAK channel to block apoptosis; in this issue Llambi et al. (2011) identify two modes of apoptosis inhibition that exhibit surprisingly different behavior upon repeat proapoptotic challenges by BH3-only proteins.

Granzyme B-dependent proteolysis acts as a switch to enhance the proinflammatory activity of IL-1α.

Granzyme B is a cytotoxic lymphocyte-derived protease that plays a central role in promoting apoptosis of virus-infected target cells, through direct proteolysis and activation of constituents of the cell death machinery. However, previous studies have also implicated granzymes A and B in the production of proinflammatory cytokines, via a mechanism that remains undefined. Here we show that IL-1α is a substrate for granzyme B and that proteolysis potently enhanced the biological activity of this cytokine in vitro as well as in vivo. Consistent with this, compared with full-length IL-1α, granzyme B-processed IL-1α exhibited more potent activity as an immunoadjuvant in vivo. Furthermore, proteolysis of IL-1α within the same region, by proteases such as calpain and elastase, was also found to enhance its biological potency. Thus, IL-1α processing by multiple immune-related proteases, including granzyme B, acts as a switch to enhance the proinflammatory properties of this cytokine.

Suppression of interleukin-33 bioactivity through proteolysis by apoptotic caspases.

Interleukin-33 (IL-33) is a member of the IL-1 family and is involved in polarization of T cells toward a T helper 2 (Th2) cell phenotype. IL-33 is thought to be activated via caspase-1-dependent proteolysis, similar to the proinflammatory cytokines IL-1 beta and IL-18, but this remains unproven. Here we showed that IL-33 was processed by caspases activated during apoptosis (caspase-3 and -7) but was not a physiological substrate for caspases associated with inflammation (caspase-1, -4, and -5). Furthermore, caspase-dependent processing of IL-33 was not required for ST2 receptor binding or ST2-dependent activation of the NF-kappaB transcription factor. Indeed, caspase-dependent proteolysis of IL-33 dramatically attenuated IL-33 bioactivity in vitro and in vivo. These data suggest that IL-33 does not require proteolysis for activation, but rather, that IL-33 bioactivity is diminished through caspase-dependent proteolysis within apoptotic cells. Thus, caspase-mediated proteolysis acts as a switch to dampen the proinflammatory properties of IL-33.

Fas and TRAIL 'death receptors' as initiators of inflammation: Implications for cancer.

Fas (CD95/APO-1) and TRAIL (CD253, TNFSF10, APO2) are members of a subset of the TNF receptor superfamily known as 'death receptors'. To date, the overwhelming majority of studies on Fas and TRAIL (TNF-related apoptosis-inducing ligand) have explored the role of these receptors as initiators of apoptosis. However, sporadic reports also suggest that engagement of the Fas and TRAIL receptors can lead to other outcomes such as cytokine and chemokine production, cell proliferation, cell migration and differentiation. Indeed, although transformed cells frequently express Fas and TRAIL, most do not undergo apoptosis upon engagement of these receptors and significant effort has been devoted toward exploring how to sensitize such cells to the pro-apoptotic effects of 'death receptor' stimulation. Moreover, the expression of Fas and TRAIL receptors is greatly elevated in many cancer types such as hepatocellular carcinoma, renal carcinoma and ovarian cancer, suggesting that such tumors benefit from the expression of these receptors. Furthermore, several studies have shown that tumor proliferation, progression and invasion can be impaired through blocking or downregulation of Fas expression, but the mechanistic basis for these effects is largely unknown. Thus, the characterization of Fas and TRAIL as 'death receptors' is a gross oversimplification, especially in the context of cancer. It is becoming increasingly clear that 'death receptor' engagement can lead to outcomes, other than apoptosis, that become subverted by certain tumors to their benefit. Here we will discuss death-independent outcomes of Fas and TRAIL signaling and their implications for cancer.

Emerging role for members of the Bcl-2 family in mitochondrial morphogenesis.

Bcl-2 family proteins regulate apoptosis by controlling the release of mitochondrial cytochrome c via the Bax/Bak channel. However, recent studies have also implicated several members of this family in the regulation of mitochondrial fission/fusion dynamics. It has been debated whether the role of Bcl-2 proteins in mitochondrial morphogenesis is functionally distinct from their role in apoptosis, with some arguing that Bax/Bak-induced mitochondrial fission promotes apoptosis-associated cytochrome c release, while others suggest that these functions are separable. Here we review this emerging area and argue for a role for the Bcl-2 family as novel regulators of mitochondrial morphogenesis.

Bax- or Bak-induced mitochondrial fission can be uncoupled from cytochrome C release.

Bax and Bak promote apoptosis by perturbing the permeability of the mitochondrial outer membrane and facilitating the release of cytochrome c by a mechanism that is still poorly defined. During apoptosis, Bax and Bak also promote fragmentation of the mitochondrial network, possibly by activating the mitochondrial fission machinery. It has been proposed that Bax/Bak-induced mitochondrial fission may be required for release of cytochrome c from the mitochondrial intermembrane space, although this has been a subject of debate. Here we show that Bcl-xL, as well as other members of the apoptosis-inhibitory subset of the Bcl-2 family, antagonized Bax and/or Bak-induced cytochrome c release but failed to block mitochondrial fragmentation associated with Bax/Bak activation. These data suggest that Bax/Bak-initiated remodeling of mitochondrial networks and cytochrome c release are separable events and that Bcl-2 family proteins can influence mitochondrial fission-fusion dynamics independent of apoptosis.

Inhibitor of apoptosis proteins (IAPs) and their antagonists regulate spontaneous and tumor necrosis factor (TNF)-induced proinflammatory cytokine and chemokine production.

Inhibitor of apoptosis proteins (IAPs) play a major role in determining whether cells undergo apoptosis in response to TNF as well as other stimuli. However, TNF is also highly proinflammatory through its ability to trigger the secretion of multiple inflammatory cytokines and chemokines, which is arguably the most important role of TNF in vivo. Indeed, deregulated production of TNF-induced cytokines is a major driver of inflammation in several autoimmune conditions such as rheumatoid arthritis. Here, we show that IAPs are required for the production of multiple TNF-induced proinflammatory mediators. Ablation or antagonism of IAPs potently suppressed TNF- or RIPK1-induced proinflammatory cytokine and chemokine production. Surprisingly, IAP antagonism also led to spontaneous production of chemokines, particularly RANTES, in vitro and in vivo. Thus, IAPs play a major role in influencing the production of multiple inflammatory mediators, arguing that these proteins are important regulators of inflammation in addition to apoptosis. Furthermore, small molecule IAP antagonists can modulate spontaneous as well as TNF-induced inflammatory responses, which may have implications for use of these agents in therapeutic settings.