Apoptosis is fundamental in maintaining cell balance in multicellular organisms, and caspases play a crucial role in apoptosis pathways. It is reported that apoptosis plays an important role in tick salivary gland degeneration. Several different caspases have been found in ticks, but the interactions between them are currently unknown. Here, we report three new caspases, isolated from the salivary glands of the tick Rhipicephalus haemaphysaloides.

The balance of pro-apoptotic and pro-survival proteins defines a cell's fate. These processes are controlled through an interdependent and finely tuned protein network that enables survival or leads to apoptotic cell death. The caspase family of proteases is central to this apoptotic network, with initiator and executioner caspases, and their interaction partners, regulating and executing apoptosis. In this work, we interrogate and modulate this network by exogenously introducing specific initiator or executioner caspase proteins. Each caspase is exogenously introduced using redox-responsive polymeric nanogels. Although caspase-3 might be expected to be the most effective due to the centrality of its role in apoptosis and its heightened catalytic efficiency relative to other family members, we observed that caspase-7 and caspase-9 are the most effective at inducing apoptotic cell death. By critically analyzing the introduced activity of the delivered caspase, the pattern of substrate cleavage, as well as the ability to activate endogenous caspases, we conclude that the efficacy of each caspase correlated with the levels of pro-survival factors that both directly and indirectly impact the introduced caspase. These findings lay the groundwork for developing methods for exogenous introduction of caspases as a therapeutic option that can be tuned to the apoptotic balance in a proliferating cell.

Heterozygosity for mutations in ribosomal protein genes frequently leads to a dominant phenotype of retarded growth and small adult bristles in Drosophila (the Minute phenotype). Cells with Minute genotypes are subject to cell competition, characterized by their selective apoptosis and removal in mosaic tissues that contain wild-type cells. Competitive apoptosis was found to depend on the pro-apoptotic reaper, grim and head involution defective genes but was independent of p53. Rp/+ cells are protected by anti-apoptotic baculovirus p35 expression but lacked the usual hallmarks of 'undead' cells. They lacked Dronc activity, and neither expression of dominant-negative Dronc nor dronc knockdown by dsRNA prevented competitive apoptosis, which also continued in dronc null mutant cells or in the absence of the initiator caspases dredd and dream/strica. Only simultaneous knockdown of dronc and dream/strica by dsRNA was sufficient to protect Rp/+ cells from competition. By contrast, Rp/Rp cells were also protected by baculovirus p35, but Rp/Rp death was dronc-dependent, and undead Rp/Rp cells exhibited typical dronc-dependent expression of Wingless. Independence of p53 and unusual dependence on Dream/Strica distinguish competitive cell death from noncompetitive apoptosis of Rp/Rp cells and from many other examples of cell death.

Initiator caspases are important components of cellular apoptotic signaling and they can activate effector caspases in extrinsic and intrinsic apoptotic pathways. The simultaneous detection of multiple initiator caspases is essential for apoptosis mechanism studies and disease therapy. Herein, we develop a sensitive nanosensor based on the integration of exonuclease III (Exo III)-powered three-dimensional (3D) DNA walker with single-molecule detection for the simultaneous measurement of initiator caspase-8 and caspase-9. This assay involves two peptide-DNA detection probe-conjugated magnetic beads and two signal probe-conjugated gold nanoparticles (signal probes@AuNPs). The presence of caspase-8 and caspase-9 can induce the cleavage of peptides in two peptide-DNA detection probes, releasing two trigger DNAs from the magnetic beads, respectively. The two trigger DNAs can serve as the walker DNA to walk on the surface of the signal probes@AuNPs powered by Exo III digestion, liberating numerous Cy5 and Texas Red fluorophores which can be quantified by single-molecule detection, with Cy5 indicating caspase-8 and Texas Red indicating caspase-9. Notably, the introduction of the AuNP-based 3D DNA walker greatly reduces the background signal and amplifies the output signals, and the introduction of single-molecule detection further improves the detection sensitivity. This nanosensor is very sensitive with a detection limit of 2.08 × 10-6 U μL-1 for caspase-8 and 1.71 × 10-6 U μL-1 for caspase-9, and it can be used for the simultaneous screening of caspase inhibitors and the measurement of endogenous caspase activity in various cell lines at the single-cell level. Moreover, this nanosensor can be extended to detect various proteases by simply changing the peptide sequences of the detection probes.

Clinical trials examining neuroprotective strategies after brain injury, including those targeting cell death mechanisms, have been underwhelming. This may be in part due to an incomplete understanding of the signalling mechanisms that induce cell death after traumatic brain injury. The recent identification of a new family of death receptors that initiate pro-cell death signals in the absence of their ligand, called dependence receptors, provides new insight into the factors that contribute to brain injury. Here, we show that blocking the dependence receptor signalling of EphB3 improves oligodendrocyte cell survival in a murine controlled cortical impact injury model, which leads to improved myelin sparing, axonal conductance and behavioural recovery. EphB3 also functions as a cysteine-aspartic protease substrate, where the recruitment of injury-dependent adaptor protein Dral/FHL-2 together with capsase-8 or -9 leads to EphB3 cleavage to initiate cell death signals in murine and human traumatic brain-injured patients, supporting a conserved mechanism of cell death. These pro-apoptotic responses can be blocked via exogenous ephrinB3 ligand administration leading to improved oligodendrocyte survival. In short, our findings identify a novel mechanism of oligodendrocyte cell death in the traumatically injured brain that may reflect an important neuroprotective strategy in patients.

The cleavage of nuclear proteins by caspases promotes nuclear breakdown and, therefore, plays a key role in apoptosis execution. However, the detailed molecular mechanisms of these events remain unclear. To get more insights into the mechanisms of nuclear events during apoptosis we set up a rapid fractionation protocol for the separation of the cytoplasmic and nuclear fractions of cells undergoing cisplatin-induced apoptosis. Importantly, nuclear accumulation of effector caspase-3 as well as initiator caspase-2, -8 and -9 was observed using the developed protocol and immunofluorescence microscopy. The detection of caspases and their cleavage products in the nucleus occurred within the same time interval after cisplatin treatment and took place shortly before nuclear fragmentation. The entry of initiator caspases to the nucleus was independent of caspase-3. Given that all three initiator caspases had catalytic activity in the nuclei, our findings indicate that initiator caspases might participate in the proteolysis of nuclear components during apoptosis, promoting its disintegration and apoptotic cell death.

Coral reefs are experiencing precipitous declines around the globe with coral diseases and temperature-induced bleaching being primary drivers of these declines. Regulation of apoptotic cell death is an important component in the coral stress response. Although cnidaria are known to contain complex apoptotic signaling pathways, similar to those in vertebrates, the mechanisms leading to cell death are largely unexplored. We identified and characterized two caspases each from Orbicella faveolata, a disease-sensitive reef-building coral, and Porites astreoides, a disease-resistant reef-building coral. The caspases are predicted homologs of the human executioner caspases-3 and -7, but OfCasp3a (Orbicella faveolata caspase-3a) and PaCasp7a (Porites astreoides caspase-7a), which we show to be DXXDases, contain an N-terminal caspase activation/recruitment domain (CARD) similar to human initiator/inflammatory caspases. OfCasp3b (Orbicella faveolata caspase-3b) and PaCasp3 (Porites astreoides caspase-3), which we show to be VXXDases, have short pro-domains, like human executioner caspases. Our biochemical analyses suggest a mechanism in coral which differs from that of humans, where the CARD-containing DXXDase is activated on death platforms but the protease does not directly activate the VXXDase. The first X-ray crystal structure of a coral caspase, of PaCasp7a determined at 1.57 Å resolution, reveals a conserved fold and an N-terminal peptide bound near the active site that may serve as a regulatory exosite. The binding pocket has been observed in initiator caspases of other species. These results suggest mechanisms for the evolution of substrate selection while maintaining common activation mechanisms of CARD-mediated dimerization.

Caspases are best characterized for their function in apoptosis. However, they also have non-apoptotic functions such as apoptosis-induced proliferation (AiP), where caspases release mitogens for compensatory proliferation independently of their apoptotic role. Here, we report that the unconventional myosin, Myo1D, which is known for its involvement in left/right development, is an important mediator of AiP in Drosophila. Mechanistically, Myo1D translocates the initiator caspase Dronc to the basal side of the plasma membrane of epithelial cells where Dronc promotes the activation of the NADPH-oxidase Duox for reactive oxygen species generation and AiP in a non-apoptotic manner. We propose that the basal side of the plasma membrane constitutes a non-apoptotic compartment for caspases. Finally, Myo1D promotes tumor growth and invasiveness of the neoplastic scrib RasV12 model. Together, we identified a new function of Myo1D for AiP and tumorigenesis, and reveal a mechanism by which cells sequester apoptotic caspases in a non-apoptotic compartment at the plasma membrane.

Self-digestion of cytoplasmic components is the hallmark of autophagic programmed cell death. This auto-degradation appears to be distinct from what occurs in apoptotic cells that are engulfed and digested by phagocytes. Although much is known about apoptosis, far less is known about the mechanisms that regulate autophagic cell death. Here we show that autophagic cell death is regulated by steroid activation of caspases in Drosophila salivary glands. Salivary glands exhibit some morphological changes that are similar to apoptotic cells, including fragmentation of the cytoplasm, but do not appear to use phagocytes in their degradation. Changes in the levels and localization of filamentous Actin, alpha-Tubulin, alpha-Spectrin and nuclear Lamins precede salivary gland destruction, and coincide with increased levels of active Caspase 3 and a cleaved form of nuclear Lamin. Mutations in the steroid-regulated genes beta FTZ-F1, E93, BR-C and E74A that prevent salivary gland cell death possess altered levels and localization of filamentous Actin, alpha-Tubulin, alpha-Spectrin, nuclear Lamins and active Caspase 3. Inhibition of caspases, by expression of either the caspase inhibitor p35 or a dominant-negative form of the initiator caspase Dronc, is sufficient to inhibit salivary gland cell death, and prevent changes in nuclear Lamins and alpha-Tubulin, but not to prevent the reorganization of filamentous Actin. These studies suggest that aspects of the cytoskeleton may be required for changes in dying salivary glands. Furthermore, caspases are not only used during apoptosis, but also function in the regulation of autophagic cell death.

Sf9, a cell line derived from Spodoptera frugiperda, is an ideal model organism for studying insect apoptosis. The first notable study that attempted to identify the apoptotic pathway in Sf9 was performed in 1997 and included the discovery of Sf-caspase-1, an effector caspase of Sf9. However, it was not until 2013 that the first initiator caspase in Sf9, SfDronc, was discovered, and the apoptotic pathway in Sf9 became clearer. In this study, we report another caspase of Sf9, SfDredd. SfDredd is highly similar to insect initiator caspase Dredd homologs. Experimentally, recombinant SfDredd underwent autocleavage and exhibited different efficiencies in cleavage of synthetic caspase substrates. This was attributed to its caspase activity for the predicted active site mutation blocked the above autocleavage and synthetic caspase substrates cleavage activity. SfDredd was capable of not only cleaving Sf-caspase-1 in vitro but also cleaving Sf-caspase-1 and inducing apoptosis when it was co-expressed with Sf-caspase-1 in Sf9 cells. The protein level of SfDredd was increased when Sf9 cells were treated by Actinomycin D, whereas silencing of SfDredd reduced apoptosis and Sf-caspase-1 cleavage induced by Actinomycin D treatment. These results clearly indicate that SfDredd functioned as an apoptotic initiator caspase. Apoptosis induced in Sf9 cells by overexpression of SfDredd alone was not as obvious as that induced by SfDronc alone, and the cleavage sites of Sf-caspase-1 for SfDredd and SfDronc are different. In addition, despite sharing a sequence homology with initiator caspases and possessing weak activity on initiator caspase substrates, SfDredd showed strong activity on effector caspase substrates, making it the only insect caspase reported so far functioning similar to human caspase-2 in this aspect. We believe that the discovery of SfDredd, and its different properties from SfDronc, will improve the understanding of apoptosis pathway in Sf9 cells.

Persistent cell shrinkage is a major hallmark of apoptotic cell death. The early-phase shrinkage, which starts within 30-120 min after apoptotic stimulation and is called apoptotic volume decrease (AVD), is known to be accomplished by activation of K+ channels and volume-sensitive outwardly rectifying (VSOR) Cl- channels in a manner independent of caspase-3 activation. However, it is controversial whether AVD depends on apoptotic dysfunction of mitochondria and activation of initiator caspases. Here, we observed that AVD is induced not only by a mitochondrial apoptosis inducer, staurosporine (STS), in mouse B lymphoma WEHI-231 cells, but also by ligation of the death receptor Fas in human B lymphoblastoid SKW6.4 cells, which undergo Fas-mediated apoptosis without involving mitochondria. Overexpression of Bcl-2 failed to inhibit the STS-induced AVD in WEHI-231 cells. These results indicate that AVD does not require the mitochondrial pathway of apoptosis. In human epithelial HeLa cells stimulated with anti-Fas antibody or STS, the AVD induction was found to precede activation of caspase-8 and caspase-9 and to be resistant to pan-caspase blockers. Thus, it is concluded that the AVD induction is an early event independent of the mitochondrial apoptotic signaling pathway and initiator caspase activation.

Apoptotic caspases evolved with metazoans more than 950 million years ago (MYA), and a series of gene duplications resulted in two subfamilies consisting of initiator and effector caspases. The effector caspase genes (caspases-3, -6, and -7) were subsequently fixed into the Chordata phylum more than 650 MYA when the gene for a common ancestor (CA) duplicated, and the three effector caspases have persisted throughout mammalian evolution. All caspases prefer an aspartate residue at the P1 position of substrates, so each caspase evolved discrete cellular roles through changes in substrate recognition at the P4 position combined with allosteric regulation. We examined the evolution of substrate specificity in caspase-6, which prefers valine at the P4 residue, compared with caspases-3 and -7, which prefer aspartate, by reconstructing the CA of effector caspases (AncCP-Ef1) and the CA of caspase-6 (AncCP-6An). We show that AncCP-Ef1 is a promiscuous enzyme with little distinction between Asp, Val, or Leu at P4. The specificity of caspase-6 was defined early in its evolution, where AncCP-6An demonstrates a preference for Val over Asp at P4. Structures of AncCP-Ef1 and of AncCP-6An show a network of charged amino acids near the S4 pocket that, when combined with repositioning a flexible active site loop, resulted in a more hydrophobic binding pocket in AncCP-6An. The ancestral protein reconstructions show that the caspase-hemoglobinase fold has been conserved for over 650 million years and that only three substitutions in the scaffold are necessary to shift substrate selection toward Val over Asp.

Caspases perform critical functions in both living and dying cells; however, how caspases perform physiological functions without killing the cell remains unclear. Here we identify a novel physiological function of caspases at the cortex of Drosophila salivary glands. In living glands, activation of the initiator caspase dronc triggers cortical F-actin dismantling, enabling the glands to stretch as they accumulate secreted products in the lumen. We demonstrate that tango7, not the canonical Apaf-1-adaptor dark, regulates dronc activity at the cortex; in contrast, dark is required for cytoplasmic activity of dronc during salivary gland death. Therefore, tango7 and dark define distinct subcellular domains of caspase activity. Furthermore, tango7-dependent cortical dronc activity is initiated by a sublethal pulse of the inhibitor of apoptosis protein (IAP) antagonist reaper. Our results support a model in which biological outcomes of caspase activation are regulated by differential amplification of IAP antagonists, unique caspase adaptor proteins, and mutually exclusive subcellular domains of caspase activity.Caspases are known for their role in cell death, but they can also participate in other physiological functions without killing the cells. Here the authors show that unique caspase adaptor proteins can regulate caspase activity within mutually-exclusive and independently regulated subcellular domains.

Tumor necrosis factor-related apoptosis- inducing ligand (TRAIL) -induced apoptosis, in transformed human breast epithelial MCF-7 cells, resulted in a time-dependent activation of the initiator caspases-8 and -9 and the effector caspase-7. Cleavage of caspase-8 and its preferred substrate, Bid, preceded processing of caspases-7 and -9, indicating that caspase-8 is the apical initiator caspase in TRAIL-induced apoptosis. Using transient transfection of COOH-terminal-tagged green fluorescent protein fusion constructs, caspases-3, -7, and -8 were localized throughout the cytoplasm of MCF-7 cells. TRAIL-induced apoptosis resulted in activation of caspases-3 and -7, and the redistribution of most of their detectable catalytically active small subunits into large spheroidal cytoplasmic inclusions, which lacked a limiting membrane. These inclusions, which were also induced in untransfected cells, contained cytokeratins 8, 18, and 19, together with both a phosphorylated form and a caspase-cleavage fragment of cytokeratin 18. Similarly, in untransfected breast HBL100 and lung A549 epithelial cells, TRAIL induced the formation of cytoplasmic inclusions that contained cleaved cytokeratin 18 and colocalized with active endogenous caspase-3. We propose that effector caspase-mediated cleavage of cytokeratins, resulting in disassembly of the cytoskeleton and formation of cytoplasmic inclusions, may be a characteristic feature of epithelial cell apoptosis.

Clinical observations suggest that ursodeoxycholate (UDCA) may protect from hepatocellular carcinoma in cirrhotic patients. Increased apoptosis of malignant cells is a candidate mechanism. Decreased apoptosis of cholangiocytes is proposed as a mechanism for the favourable effect of UDCA in primary biliary cirrhosis. We therefore studied the effects of different concentrations of UDCA on HepG2 cell proliferation, apoptosis and caspases activities. Apoptotic features and activities of the effector or initiator caspases-8, -9, -3 and -2 after treatment of HepG2 cells with different concentrations of UDCA alone or in combination with TNF-alpha were examined. Apoptosis was detected by DNA fragmentation and flow cytometric determination of apoptotic cells with Annexin-V/PI. UDCA significantly inhibits cell proliferation only at high concentrations, but increases apoptosis at low concentrations and protects from apoptosis at higher concentrations. TNF-alpha induced DNA fragmentation is potentiated by UDCA, but flow cytometry indicates protection from early apoptosis and increase in cell survival by low and intermediate UDCA concentrations. UDCA differentially activates initiator and effector caspases in different concentrations. These data demonstrate that the effect of UDCA on caspase activation and apoptosis of HepG2 cells is concentration-dependent and activation of the caspase cascade is not always translated into increased apoptosis. Serum levels of UDCA should be possibly monitored and dosage of the drug adjusted according to the required effect.

A great number of obsolete larval neurons in the Drosophila central nervous system are eliminated by developmentally programmed cell death (PCD) during early metamorphosis. To elucidate the mechanisms of neuronal PCD occurring during this period, we undertook genetic dissection of seven currently known Drosophila caspases in the PCD of a group of interneurons (vCrz) that produce corazonin (Crz) neuropeptide in the ventral nerve cord. The molecular death program in the vCrz neurons initiates within 1 hour after pupariation, as demonstrated by the cytological signs of cell death and caspase activation. PCD was significantly suppressed in dronc-null mutants, but not in null mutants of either dredd or strica. A double mutation lacking both dronc and strica impaired PCD phenotype more severely than did a dronc mutation alone, but comparably to a triple dredd/strica/dronc mutation, indicating that dronc is a main initiator caspase, while strica plays a minor role that overlaps with dronc's. As for effector caspases, vCrz PCD requires both ice and dcp-1 functions, as they work cooperatively for a timely removal of the vCrz neurons. Interestingly, the activation of the Ice and Dcp-1 is not solely dependent on Dronc and Strica, implying an alternative pathway to activate the effectors. Two remaining effector caspase genes, decay and damm, found no apparent functions in the neuronal PCD, at least during early metamorphosis. Overall, our work revealed that vCrz PCD utilizes dronc, strica, dcp-1, and ice wherein the activation of Ice and Dcp-1 requires a novel pathway in addition to the initiator caspases.

Caspases provide vital links in non-apoptotic regulatory networks controlling inflammation, compensatory proliferation, morphology and cell migration. How caspases are activated under non-apoptotic conditions and process a selective set of substrates without killing the cell remain enigmatic. Here we find that the Drosophila unconventional myosin CRINKLED (CK) selectively interacts with the initiator caspase DRONC and regulates some of its non-apoptotic functions. Loss of CK in the arista, border cells or proneural clusters of the wing imaginal discs affects DRONC-dependent patterning. Our data indicate that CK acts as substrate adaptor, recruiting SHAGGY46/GSK3-β to DRONC, thereby facilitating caspase-mediated cleavage and localized modulation of kinase activity. Similarly, the mammalian CK counterpart, MYO7A, binds to and impinges on CASPASE-8, revealing a new regulatory axis affecting receptor interacting protein kinase-1 (RIPK1)>CASPASE-8 signalling. Together, our results expose a conserved role for unconventional myosins in transducing caspase-dependent regulation of kinases, allowing them to take part in specific signalling events.

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.

While circadian dysfunction and neurodegeneration are correlated, the mechanism for this is not understood. It is not known if age-dependent circadian dysfunction leads to neurodegeneration or vice-versa, and the proteins that mediate the effect remain unidentified. Here, we show that the knock-down of a regulator (spag) of the circadian kinase Dbt in circadian cells lowers Dbt levels abnormally, lengthens circadian rhythms and causes expression of activated initiator caspase (Dronc) in the optic lobes during the middle of the day or after light pulses at night. Likewise, reduced Dbt activity lengthens circadian period and causes expression of activated Dronc, and a loss-of-function mutation in Clk also leads to expression of activated Dronc in a light-dependent manner. Genetic epistasis experiments place Dbt downstream of Spag in the pathway, and Spag-dependent reductions of Dbt are shown to require the proteasome. Importantly, activated Dronc expression due to reduced Spag or Dbt activity occurs in cells that do not express the spag RNAi or dominant negative Dbt and requires PDF neuropeptide signaling from the same neurons that support behavioral rhythms. Furthermore, reduction of Dbt or Spag activity leads to Dronc-dependent Drosophila Tau cleavage and enhanced neurodegeneration produced by human Tau in a fly eye model for tauopathy. Aging flies with lowered Dbt or Spag function show markers of cell death as well as behavioral deficits and shortened lifespans, and even old wild type flies exhibit Dbt modification and activated caspase at particular times of day. These results suggest that Dbt suppresses expression of activated Dronc to prevent Tau cleavage, and that the circadian clock defects confer sensitivity to expression of activated Dronc in response to prolonged light. They establish a link between the circadian clock factors, light, cell death pathways and Tau toxicity, potentially via dysregulation of circadian neuronal remodeling in the optic lobes.

Apoptosis is an evolutionary conserved cell death mechanism, which requires activation of initiator and effector caspases. The Drosophila initiator caspase Dronc, the ortholog of mammalian Caspase-2 and Caspase-9, has an N-terminal CARD domain that recruits Dronc into the apoptosome for activation. In addition to its role in apoptosis, Dronc also has non-apoptotic functions such as compensatory proliferation. One mechanism to control the activation of Dronc is ubiquitylation. However, the mechanistic details of ubiquitylation of Dronc are less clear. For example, monomeric inactive Dronc is subject to non-degradative ubiquitylation in living cells, while ubiquitylation of active apoptosome-bound Dronc triggers its proteolytic degradation in apoptotic cells. Here, we examined the role of non-degradative ubiquitylation of Dronc in living cells in vivo, i.e. in the context of a multi-cellular organism. Our in vivo data suggest that in living cells Dronc is mono-ubiquitylated on Lys78 (K78) in its CARD domain. This ubiquitylation prevents activation of Dronc in the apoptosome and protects cells from apoptosis. Furthermore, K78 ubiquitylation plays an inhibitory role for non-apoptotic functions of Dronc. We provide evidence that not all of the non-apoptotic functions of Dronc require its catalytic activity. In conclusion, we demonstrate a mechanism whereby Dronc's apoptotic and non-apoptotic activities can be kept silenced in a non-degradative manner through a single ubiquitylation event in living cells.