Caspase-2 activation in the absence of PIDDosome formation

PIDD (p53-induced protein with a death domain [DD]), together with the bipartite adapter protein RAIDD (receptor-interacting protein-associated ICH-1/CED-3 homologous protein with a DD), is implicated in the activation of pro–caspase-2 in a high molecular weight complex called the PIDDosome during apoptosis induction after DNA damage. To investigate the role of PIDD in cell death initiation, we generated PIDD-deficient mice. Processing of caspase-2 is readily detected in the absence of PIDDosome formation in primary lymphocytes. Although caspase-2 processing is delayed in simian virus 40–immortalized pidd^−/− mouse embryonic fibroblasts, it still depends on loss of mitochondrial integrity and effector caspase activation. Consistently, apoptosis occurs normally in all cell types analyzed, suggesting alternative biological roles for caspase-2 after DNA damage. Because loss of either PIDD or its adapter molecule RAIDD did not affect subcellular localization, nuclear translocation, or caspase-2 activation in high molecular weight complexes, we suggest that at least one alternative PIDDosome-independent mechanism of caspase-2 activation exists in mammals in response to DNA damage.     

In vitro reconstitution of the interactions in the PIDDosome

Caspase-2 is critical for genotoxic stress induced apoptosis and is activated by formation of the PIDDosome, an oligomeric caspase-2 activating complex. The PIDDosome comprises three protein components, PIDD, RAIDD, and caspase-2. RAIDD contains both a death domain (DD) and a caspase recruitment domain (CARD). It acts as the bridge to recruit PIDD using the DD: DD interaction and to recruit caspase-2 via the CARD: CARD interaction. Here we report biochemical characterization and in vitro reconstitution of the core interactions in the PIDDosome. We show that RAIDD CARD and RAIDD DD interact with their binding partners, caspase-2 CARD and PIDD DD, respectively. However, full-length RAIDD fails to interact with either caspase-2 CARD or PIDD DD under a physiological buffer condition. We reveal that this lack of interaction of full-length RAIDD is not due to its tendency to aggregate under the physiological buffer condition, as decreasing full-length RAIDD aggregation using high salt or high pH is not able to promote complex formation. Instead, full-length RAIDD forms both binary and ternary complexes under a low salt condition. Successful in vitro reconstitution of the ternary complex provides a basis for further structural studies of the PIDDosome.     

mE10, a novel caspase recruitment domain-containing proapoptotic molecule

Apoptotic signaling is mediated by homophilic interactions between conserved domains present in components of the death pathway. The death domain, death effector domain, and caspase recruitment domain (CARD) are examples of such interaction motifs. We have identified a novel mammalian CARD-containing adaptor molecule termed mE10 (mammalian E10). The N-terminal CARD of mE10 exhibits significant homology (47% identity and 64% similarity) to the CARD of a gene from Equine Herpesvirus type 2. The C-terminal region is unique. Overexpression of mE10 in MCF-7 human breast carcinoma cells induces apoptosis. Mutational analysis indicates that CARD-mediated mE10 oligomerization is essential for killing activity. The C terminus of mE10 bound to the zymogen form of caspase-9 and promoted its processing to the active dimeric species. Taken together, these data suggest a model where autoproteolytic activation of pro-caspase-9 is mediated by mE10-induced oligomerization.     

Role of prodomain in importin-mediated nuclear localization and activation of caspase-2

Caspase-2 is unique among mammalian caspases because it localizes to the nucleus in a prodomain-dependent manner. The caspase-2 prodomain also regulates caspase-2 activity via a caspase recruitment domain that mediates oligomerization of procaspase-2 molecules and their subsequent autoactivation. In this study we sought to map specific functional regions in the caspase-2 prodomain that regulate its nuclear transport and also its activation. Our data indicate that caspase-2 contains a classical nuclear localization signal (NLS) at the C terminus of the prodomain which is recognized by the importin alpha/beta heterodimer. The mutation of a conserved Lys residue in the NLS abolishes nuclear localization of caspase-2 and binding to the importin alpha/beta heterodimer. Although caspase-2 is imported into the nucleus, mutants lacking the NLS were still capable of inducing apoptosis upon overexpression in transfected cells. We define a region in the prodomain that regulates the ability of caspase-2 to form dot- and filament-like structures when ectopically expressed, which in turn promotes cell killing. Our data provides a mechanism for caspase-2 nuclear import and demonstrate that association of procaspase-2 into higher order structures, rather than its nuclear localization, is required for caspase-2 activation and its ability to induce apoptosis.     

PIDD Death-Domain Phosphorylation by ATM Controls Prodeath versus Prosurvival PIDDosome Signaling

Biochemical evidence implicates the death-domain (DD) protein PIDD as a molecular switch capable of?signaling cell survival or death in response to genotoxic stress. PIDD activity is determined by binding-partner selection at its DD: whereas recruitment of RIP1 triggers prosurvival NF-κB signaling, recruitment of RAIDD activates proapoptotic caspase-2 via PIDDosome formation. However, it remains unclear how interactor selection, and thus fate decision, is regulated at the PIDD platform. We show that the PIDDosome functions in the "Chk1-suppressed" apoptotic response to DNA damage, a conserved ATM/ATR-caspase-2 pathway antagonized by Chk1. In this pathway, ATM phosphorylates PIDD on Thr788 within the DD. This phosphorylation is necessary and sufficient for RAIDD binding and caspase-2 activation. Conversely, nonphosphorylatable PIDD fails to bind RAIDD or activate caspase-2, and engages prosurvival RIP1 instead. Thus, ATM phosphorylation of the PIDD DD enables a binary switch through which cells elect to survive or die upon DNA injury.     

RAIDD is required for apoptosis of PC12 cells and sympathetic neurons induced by trophic factor withdrawal

Caspase 2 has been implicated in trophic deprivation-induced neuronal death. We have shown that overexpression of the caspase 2-binding protein RAIDD induces neuronal apoptosis, acting synergistically with trophic deprivation. Currently, we examine the role of endogenous RAIDD in apoptosis of PC12 cells and sympathetic neurons. Expression of a truncated caspase recruitment domain-only form of caspase 2, which presumably disrupts the RAIDD interaction with endogenous caspase 2, attenuated trophic deprivation-induced apoptosis. Furthermore, downregulation of RAIDD by small interfering RNA led to inhibition of trophic deprivation-induced death, whereas death induced by DNA damage, which is not caspase 2-mediated, was not inhibited. Therefore, RAIDD, likely through interaction with caspase 2, is involved in trophic deprivation-induced neuronal apoptosis. This is the first demonstration of the involvement of RAIDD in apoptosis, and provides further support for the idea that apoptotic pathways in the same system may differ depending on the initiating stimulus.     

Upon intracellular processing, the C-terminal death domain-containing fragment of the p53-inducible PIDD/LRDD protein translocates to the nucleoli and interacts with nucleolin

The p53-inducible and death domain-containing PIDD/LRDD protein has been described as an adaptor protein, which forms large protein complexes with RAIDD, another death domain-containing protein, leading to recruitment, and activation of the initiator caspase-2, and p53-mediated apoptosis. Here, we describe in further detail the proteolytic processing of PIDD/LRDD that occurs in healthy cells before induction of apoptosis. We could demonstrate that the C-terminal fragment containing the PIDD death domain shuttles into the nucleoli. This translocation is mediated by or leads to the interaction of the PIDD death domain with nucleolin, a protein important for rRNA processing within nucleoli and possibly involved in the DNA damage response. Ectopically expressed LRDD and endogenous nucleolin co-localized within the nucleoli, and overexpression of both full-length LRDD and the LRDD death domain sensitized cells for UV-induced apoptosis. When expressed alone, the PIDD/LRDD death domain tended to form large filamentous structures resembling so-called death filaments. The functional consequences of the identified PIDD/nucleolin interaction remain to be elucidated, but may be related to a recently discovered new role for PIDD in the activation of NF-kappaB upon genotoxic stress.     

Crystal structure of RAIDD death domain implicates potential mechanism of PIDDosome assembly

Caspase-2 is implicated in stress-induced apoptosis that acts as an upstream initiator of mitochondrial permeabilization. Recent studies have shown that caspase-2 activation requires a molecular complex known as the PIDDosome comprising the p53-inducible protein PIDD, the adapter protein RAIDD and caspase-2. RAIDD has an N-terminal caspase recruitment domain (CARD) that interacts with the CARD of caspase-2 and a C-terminal death domain (DD) that interacts with the DD in PIDD. As a first step towards elucidating the molecular mechanisms of caspase-2 activation, we report the crystal structure of RAIDD DD at 2.0 A resolution. The high-resolution structure reveals important features of RAIDD DD that may be important for DD folding and dynamics and for assembly of the PIDDosome.     

RAIDD aggregation facilitates apoptotic death of PC12 cells and sympathetic neurons

In human cell lines, the caspase 2 adaptor RAIDD interacts selectively with caspase 2 through its caspase recruitment domain (CARD) and leads to caspase 2-dependent death. Whether RAIDD induces such effects in neuronal cells is unknown. We have previously shown that caspase 2 is essential for apoptosis of trophic factor-deprived PC12 cells and rat sympathetic neurons. We report here that rat RAIDD, cloned from PC12 cells, interacts with rat caspase 2 CARD. RAIDD overexpression induced caspase 2 CARD- and caspase 9-dependent apoptosis of PC12 cells and sympathetic neurons. Apoptosis correlated with the formation of discrete perinuclear aggregates. Both death and aggregates required the expression of full-length RAIDD. Such aggregates may enable more effective activation of caspase 2 through close proximity. Following trophic deprivation, RAIDD overexpression increased death and aggregate formation. Therefore, RAIDD aggregation is important for its death-promoting effects and may play a role in trophic factor withdrawal-induced neuronal apoptosis.     

Neuronal caspase 2 activity and function requires RAIDD, but not PIDD

Caspase 2 was initially identified as a neuronally expressed developmentally down-regulated gene (HUGO gene nomenclature CASP2) and has been shown to be required for neuronal death induced by several stimuli, including NGF (nerve growth factor) deprivation and Aβ (β-amyloid). In non-neuronal cells the PIDDosome, composed of caspase 2 and two death adaptor proteins, PIDD (p53-inducible protein with a death domain) and RAIDD {RIP (receptor-interacting protein)-associated ICH-1 [ICE (interleukin-1β-converting enzyme)/CED-3 (cell-death determining 3) homologue 1] protein with a death domain}, has been proposed as the caspase 2 activation complex, although the absolute requirement for the PIDDosome is not clear. To investigate the requirement for the PIDDosome in caspase-2-dependent neuronal death, we have examined the necessity for each component in induction of active caspase 2 and in execution of caspase-2-dependent neuronal death. We find that both NGF deprivation and Aβ treatment of neurons induce active caspase 2 and that induction of this activity depends on expression of RAIDD, but is independent of PIDD expression. We show that treatment of wild-type or PIDD-null neurons with Aβ or NGF deprivation induces formation of a complex of caspase 2 and RAIDD. We also show that caspase-2-dependent execution of neurons requires RAIDD, not PIDD. Caspase 2 activity can be induced in neurons from PIDD-null mice, and NGF deprivation or Aβ use caspase 2 and RAIDD to execute death of these neurons.     

Caspase-2 can trigger cytochrome C release and apoptosis from the nucleus

The cysteine proteases specific for aspartic residues, known as caspases, are localized in different subcellular compartments and play specific roles during the regulative and the executive phase of the cell death process. Here we investigated the subcellular localization of caspase-2 in healthy cells and during the execution of the apoptotic program. We have found that caspase-2 is a nuclear resident protein and that its import into the nucleus is regulated by two different nuclear localization signals. We have shown that in an early phase of apoptosis caspase-2 can trigger mitochondrial dysfunction from the nucleus without relocalizing into the cytoplasm. Release of cytochrome c occurs in the absence of overt alteration of the nuclear pores and changes of the nuclear/cytoplasmic barrier. Addition of leptomycin B, an inhibitor of nuclear export, did not interfere with the ability of caspase-2 to trigger cytochrome c release. Only during the late phase of the apoptotic process can caspase-2 relocalize in the cytoplasm, as consequence of an increase in the diffusion limits of the nuclear pores. Taken together these data indicate the existence of a nuclear/mitochondrial apoptotic pathway elicited by caspase-2.     

Crystal structure of MC159 reveals molecular mechanism of DISC assembly and FLIP inhibition

The death-inducing signaling complex (DISC) comprising Fas, Fas-associated death domain (FADD), and caspase-8/10 is assembled via homotypic associations between death domains (DDs) of Fas and FADD and between death effector domains (DEDs) of FADD and caspase-8/10. Caspase-8/10 and FLICE/caspase-8 inhibitory proteins (FLIPs) that inhibit caspase activation at the DISC level contain tandem DEDs. Here, we report the crystal structure of a viral FLIP, MC159, at 1.2 Angstroms resolution. It reveals a noncanonical fold of DED1, a dumbbell-shaped structure with rigidly associated DEDs and a different mode of interaction in the DD superfamily. Whereas the conserved hydrophobic patch of DED1 interacts with DED2, the corresponding region of DED2 mediates caspase-8 recruitment and contributes to DISC assembly. In contrast, MC159 cooperatively assembles with Fas and FADD via an extensive surface that encompasses the conserved charge triad. This interaction apparently competes with FADD self-association and disrupts higher-order oligomerization required for caspase activation in the DISC.     

PIDD mediates and stabilizes the interaction between RAIDD and Caspase-2 for the PIDDosome assembly

The PIDDosome, which is an oligomeric signaling complex composed of PIDD, RAIDD and caspase-2, can induce proximity-based dimerization and activation of caspase-2. In the PIDDosome assembly, the adaptor protein RAIDD interacts with PIDD and caspase-2 via CARD:CARD and DD:DD, respectively. To analyze the PIDDosome assembly, we purified all of the DD superfamily members and performed biochemical analyses. The results revealed that caspase-2 CARD is an insoluble protein that can be solubilized by its binding partner, RAIDD CARD, but not by full-length RAIDD; this indicates that full-length RAIDD in closed states cannot interact with caspase-2 CARD. Moreover, we found that caspase-2 CARD can be solubilized and interact with full-length RAIDD in the presence of PIDD DD, indicating that PIDD DD initially binds to RAIDD, after which caspase-2 can be recruited to RAIDD via a CARD:CARD interaction. Our study will be useful in determining the order of assembly of the PIDDosome. [BMB Reports 2013; 46(9): 471-476]     

Caspase-3细胞定位信号的检测与分析

Caspase-3是凋亡过程中的重要作用蛋白。凋亡过程中,胞质定位的Caspase3被激活并进入细胞核中执行功能,但该定位变化的分子机制至今仍不清楚。分析caspase3中的细胞定位信号可以为深入了解该过程提供重要的线索。我们通过构建一系列含Caspase-3不同区段的截短突变体,与GFP融合表达,观察这些突变体在细胞中的定位,以鉴定Caspase-3中的核外运信号NES（Nuclear Export Signal）和核定位信号NLS（Nuclear Localization Signal）。Caspase-3中不存在明显的核定位信号NLS,但存在一个明显的核外运信号NES,该NES信号定位在caspase-3小亚基的C端（氨基酸220-245）。     

In situ trapping of activated initiator caspases reveals a role for caspase-2 in heat shock-induced apoptosis

Activation of 'initiator' (or 'apical') caspases-2, -8 or -9 (refs 1-3) is crucial for induction of apoptosis. These caspases function to activate executioner caspapses that, in turn, orchestrate apoptotic cell death. Here, we show that a cell-permeable, biotinylated pan-caspase inhibitor (bVAD-fmk) both inhibited and 'trapped' the apical caspase activated when apoptosis was triggered. As expected, only caspase-8 was trapped in response to ligation of death receptors, whereas only caspase-9 was trapped in response to a variety of other apoptosis-inducing agents. Caspase-2 was exclusively activated in heat shock-induced apoptosis. This activation of caspase-2 was also observed in cells protected from heat-shock-induced apoptosis by Bcl-2 or Bcl-xL. Reduced sensitivity to heat-shock-induced death was observed in caspase-2(-/-) cells. Furthermore, cells lacking the adapter molecule RAIDD failed to activate caspase-2 after heat shock treatment and showed resistance to apoptosis in this setting. This approach unambiguously identifies the apical caspase activated in response to apoptotic stimuli, and establishes caspase-2 as a proximal mediator of heat shock-induced apoptosis.     

The PYRIN-CARD protein ASC is an activating adaptor for caspase-1

The PYRIN and CARD domains are members of the six-helix bundle death domain-fold superfamily that mediates assembly of large signaling complexes in the apoptotic and inflammatory signaling pathways. Here we show that the PYRIN-CARD protein ASC functions as a caspase-1-activating adaptor. ASC interacted specifically with procaspase-1 via CARD-CARD interactions and induced its oligomerization. Consistent with these results ectopic expression of full-length ASC, but not its isolated CARD or PYRIN domain, with procaspase-1 induced activation of procaspase-1 and processing of pro-interleukin-1beta in transfected cells. Substitution of the PYRIN domain of ASC with an inducible FKBP12 oligomerization domain produced a molecule that can induce caspase-1 activation in response to stimulation with the oligomerization drug AP20187, suggesting that the PYRIN domain functions as an oligomerization domain, whereas the CARD domain functions as the effector domain in the caspase-1 activation pathway. Furthermore stable expression of an isolated CARD of ASC in THP-1 cells diminished interleukin-1beta generation in response to pro-inflammatory cytokines. These results indicate that ASC is involved in the caspase-1 signaling pathway by mediating the assembly of a caspase-1-inflammasome signaling complex in response to pro-inflammatory cytokine stimulation.     

Nuclear translocation of caspase-3 is dependent on its proteolytic activation and recognition of a substrate-like protein(s)

Caspase-3 is thought to play an important role(s) in the nuclear morphological changes that occur in apoptotic cells and many nuclear substrates for caspase-3 have been identified despite the cytoplasmic localization of procaspase-3. Therefore, whether activated caspase-3 is localized in the nuclei and how active caspase-3 has access to its nuclear targets are important and unresolved questions. Here we confirmed nuclear localizations for both caspase-3-p17 and caspase-3-p12 subunits of active caspase in apoptotic cells using subcellular fractionation analysis. We also prepared polyclonal and monoclonal antibodies specific for active caspase-3 to define the subcellular localization of active caspase-3. Immunocytochemical observations using anti-active caspase-3 antibodies showed nuclear accumulation of active caspase-3 during apoptosis. In addition, caspase-3, but not caspase-7, translocated from the cytoplasm into the nucleus after induction of apoptosis. Mutations at the cleavage site between the p17 and p12 subunits and the substrate recognition site for the P3 amino acid of the DXXD substrate cleavage motif inhibited nuclear translocation of caspase-3, indicating that nuclear transport of active caspase-3 required proteolytic activation and substrate recognition. These results suggest that active caspase-3 is translocated in association with a substrate-like protein(s) from the cytoplasm into the nucleus during progression through apoptosis.     

Caspase-2 activation is redundant during seizure-induced neuronal death

Seizure-induced neuronal death may be under the control of the caspase family of cell death proteases. We examined the role of caspase-2 in a model of focally evoked limbic seizures with continuous EEG recording. Seizures were elicited by microinjection of kainic acid into the amygdala of the rat and terminated after 40 min by diazepam. Caspase-2 was constitutively present in brain, mostly within neurons, and was detected in both cytoplasm and nucleus. Cleaved caspase-2 (12 kDa) was detected immediately following seizure termination within injured ipsilateral hippocampus, contiguous with increased Val-Asp-Val-Ala-Asp (VDVADase) activity, a putative measure of activated caspase-2. Expression of receptor interacting protein (RIP)-associated Ich-1-homologous protein with death domain (RAIDD) was increased following seizures, whereas expression of RIP and tumor necrosis factor receptor associated protein with death domain (TRADD), other components thought to be linked to the caspase-2 activation and signaling mechanism, were unchanged. Intracerebroventricular administration of z-VDVAD-fluoromethyl ketone blocked seizure-induced caspase-2 activity but did not alter caspase-8 activity and failed to affect DNA fragmentation or neuronal death. These data support activation of caspase-2 following seizures but suggest that parallel caspase pathways may circumvent deficits in caspase-2 function to complete the cell death process.