SfDronc,an initiator caspase involved in apoptosis in the fall armyworm Spodoptera frugiperda

Initiator caspases are the first caspases that are activated following an apoptotic stimulus, and are responsible for cleaving and activating downstream effector caspases, which directly cause apoptosis. We have cloned a cDNA encoding an ortholog of the initiator caspase Dronc in the lepidopteran insect Spodoptera frugiperda. The SfDronc cDNA encodes a predicted protein of 447 amino acids with a molecular weight of 51 kDa. Overexpression of SfDronc induced apoptosis in Sf9 cells, while partial silencing of SfDronc expression in Sf9 cells reduced apoptosis induced by baculovirus infection or by treatment with UV or actinomycin D. Recombinant SfDronc exhibited several expected biochemical characteristics of an apoptotic initiator caspase: 1) SfDronc efficiently cleaved synthetic initiator caspase substrates, but had very little activity against effector caspase substrates; 2) mutation of a predicted cleavage site at position D340 blocked autoprocessing of recombinant SfDronc and reduced enzyme activity by approximately 10-fold; 3) SfDronc cleaved the effector caspase Sf-caspase-1 at the expected cleavage site, resulting in Sf-caspase-1 activation; and 4) SfDronc was strongly inhibited by the baculovirus caspase inhibitor SpliP49, but not by the related protein AcP35. These results indicate that SfDronc is an initiator caspase involved in caspase-dependent apoptosis in S. frugiperda, and as such is likely to be responsible for the initiator caspase activity in S. frugiperda cells known as Sf-caspase-X.     

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.     

Induction of apoptosis in SF21 cell line by conditioned medium of the entomopathogenic fungus, Nomuraea rileyi, through Sf-caspase-1 signaling pathway

The apoptosis in SF-21 cell line can be induced by the conditioned medium (CM) of the entomopathogenic fungus, Nomuraea rileyi, based on changes in morphology and formation of apoptotic bodies in cultured cells, and with the onset of DNA fragmentation as shown by TUNEL staining and agarose electrophoresis. Moreover, the induction of apoptosis in SF-21 cells was inhibited by adding the inhibitor of effector caspase, viz. z-DEVD-fmk, to the CM, indicating that Sf-caspase-1 is involved in this apoptosis. Similarly, the inhibitor of initiator caspase, viz., z-VAD-fmk, inhibited apoptosis. Therefore, both initiator and effector caspases are possibly involved in the apoptosis of SF-21 cells. In addition, we detected Sf-caspase-1 activity in the process of apoptosis in SF-21 cells, suggesting that the effector caspase in SF-21 is similar to that found in mammalian cells. Our results also indicated that the apoptosis found in this line is accomplished through a Sf-caspase-1 signaling pathway.     

Apoptosis in motion. An apical, P35-insensitive caspase mediates programmed cell death in insect cells

Activation of caspases by proteolytic processing is a critical step during apoptosis in metazoans. Here we use high resolution time lapse microscopy to show a tight link between caspase activation and the morphological events delineating apoptosis in cultured SF21 cells from the moth Spodoptera frugiperda, a model insect system. The principal effector caspase, Sf-caspase-1, is proteolytically activated during SF21 apoptosis. To define the potential role of initiator caspases in vivo, we tested the effect of cell-permeable peptide inhibitors on pro-Sf-caspase-1 processing. Anti-caspase peptide analogues prevented apoptosis induced by diverse signals, including UV radiation and baculovirus infection. IETD-fmk potently inhibited the initial processing of pro-Sf-caspase-1 at the junction (TETD-G) of the large and small subunit, a cleavage that is blocked by inhibitor of apoptosis Op-IAP but not pancaspase inhibitor P35. Because Sf-caspase-1 was inhibited poorly by IETD-CHO, our data indicated that the protease responsible for the first step in pro-Sf-caspase-1 activation is a distinct apical caspase. Thus, Sf-caspase-1 activation is mediated by a novel, P35-resistant caspase. These findings support the hypothesis that apoptosis in insects, like that in mammals, involves a cascade of caspase activations.     

Silkworm hemolymph as a potent inhibitor of apoptosis in Sf9 cells

We have previously shown that silkworm hemolymph exhibits anti-apoptotic activity against baculovirus-induced Sf9 cell apoptosis. In this study, using various chemical inducers, such as actinomycin D, camptothecin, and staurosporine, we found that silkworm hemolymph inhibits insect cell apoptosis induced not only by baculovirus but also by chemical inducers. This indicates that silkworm hemolymph contains anti-apoptotic components that work directly in insect cell apoptosis without any booster expression of baculoviral genes. With the analysis of Sf-caspase-1 activity, it was found that the inhibitory effect of silkworm hemolymph works in a further upstream step than the Sf-caspase-1 activation step.     

Reactive-site cleavage residues confer target specificity to baculovirus P49, a dimeric member of the P35 family of caspase inhibitors

Baculovirus proteins P49 and P35 are potent suppressors of apoptosis in diverse organisms. Although related, P49 and P35 inhibit initiator and effector caspases, respectively, during infection of permissive insect cells. The molecular basis of this novel caspase specificity is unknown. To advance strategies for selective inhibition of the cell death caspases, we investigated biochemical differences between these baculovirus substrate inhibitors. We report here that P49 and P35 use similar mechanisms for stoichiometric inhibition that require caspase cleavage of their reactive site loops (RSL) and chemical contributions of a conserved N-terminal cysteine to stabilize the resulting inhibitory complex. Our data indicated that P49 functions as a homodimer that simultaneously binds two caspases. In contrast, P35 is a monomeric, monovalent inhibitor. P49 and P35 also differ in their RSL caspase recognition sequences. We tested the role of the P₄-P₁ recognition motif for caspase specificity by monitoring virus-induced proteolytic processing of Sf-caspase-1, the principal effector caspase of the host insect Spodoptera frugiperda. When P49's TVTD recognition motif was replaced with P35's DQMD motif, P49 was impaired for inhibition of the initiator caspase that cleaves and activates pro-Sf-caspase-1 and instead formed a stable inhibitory complex with active Sf-caspase-1. In contrast, the effector caspase specificity of P35 was unaltered when P35's DQMD motif was replaced with TVTD. We concluded that the TVTD recognition motif is required but not sufficient for initiator caspase inhibition by P49. Our findings demonstrate a critical role for the P₄-P₁ recognition site in caspase specificity by P49 and P35 and indicate that additional determinants are involved in target selection.     

喜树碱诱导的草地贪夜蛾Sf9细胞凋亡

传统植物源杀虫剂喜树碱具有优异的抑制昆虫生长发育活性, 其诱导昆虫细胞凋亡的作用方式和机制尚不明确, 极大地限制了喜树碱在植物保护领域的应用开发。本研究以1 μmol/L喜树碱诱导草地贪夜蛾Spodoptera frugiperda Sf9细胞呈现细胞皱缩、微绒毛消失和染色质边集等典型细胞凋亡早期超微结构形态特征, 中期凋亡小体逐渐出现并急剧增多, DNA电泳分析可见清晰DNA片段化凋亡特征。流式细胞术分析表明1 μmol/L喜树碱诱导Sf9细胞12 h凋亡率达到最大值39.67%, 是对照的13.13倍, 随后减小。喜树碱诱导Sf9细胞凋亡在12 h和24 h 时Sf caspase-1分别出现两个活性高峰, 表明其作为效应因子在细胞凋亡级联反应过程中具有影响作用。喜树碱显著抑制Sf9细胞拓扑异构酶Ⅰ活性, 阻断解旋负超螺旋pBR322 DNA, 导致DNA损伤进而启动细胞凋亡级联反应使Sf caspase-1活性增加, 提示其信号转导过程是细胞凋亡诱导机制之一。本研究通过分析喜树碱的诱导昆虫Sf9细胞凋亡, 对揭示喜树碱诱导昆虫细胞凋亡的作用机制具有重要启示和帮助。     

Caspase 3 attenuates XIAP (X-linked inhibitor of apoptosis protein)-mediated inhibition of caspase 9

During apoptosis, the initiator caspase 9 is activated at the apoptosome after which it activates the executioner caspases 3 and 7 by proteolysis. During this process, caspase 9 is cleaved by caspase 3 at Asp(330), and it is often inferred that this proteolytic event represents a feedback amplification loop to accelerate apoptosis. However, there is substantial evidence that proteolysis per se does not activate caspase 9, so an alternative mechanism for amplification must be considered. Cleavage at Asp(330) removes a short peptide motif that allows caspase 9 to interact with IAPs (inhibitors of apoptotic proteases), and this event may control the amplification process. We show that, under physiologically relevant conditions, caspase 3, but not caspase 7, can cleave caspase 9, and this does not result in the activation of caspase 9. An IAP antagonist disrupts the inhibitory interaction between XIAP (X-linked IAP) and caspase 9, thereby enhancing activity. We demonstrate that the N-terminal peptide of caspase 9 exposed upon cleavage at Asp330 cannot bind XIAP, whereas the peptide generated by autolytic cleavage of caspase 9 at Asp315 binds XIAP with substantial affinity. Consistent with this, we found that XIAP antagonists were only capable of promoting the activity of caspase 9 when it was cleaved at Asp315, suggesting that only this form is regulated by XIAP. Our results demonstrate that cleavage by caspase 3 does not activate caspase 9, but enhances apoptosis by alleviating XIAP inhibition of the apical caspase.     

Novel Apoptosis Suppressor Apsup from the Baculovirus Lymantria dispar Multiple Nucleopolyhedrovirus Precludes Apoptosis by Preventing Proteolytic Processing of Initiator Caspase Dronc

We previously identified a novel baculovirus-encoded apoptosis suppressor, Apsup, from the baculovirus Lymantria dispar multiple nucleopolyhedrovirus (LdMNPV). Apsup inhibits the apoptosis of L. dispar Ld652Y cells triggered by infection with p35-defective Autographa californica MNPV (vAcΔp35) and exposure to actinomycin D or UV light. Here, we examined the functional role of Apsup in apoptosis regulation in insect cells. Apsup prevented apoptosis and the proteolytic processing of L. dispar initiator caspase Dronc (Ld-Dronc) in Ld652Y cells triggered by overexpression of Ld-Dronc, LdMNPV inhibitor-of-apoptosis 3 (IAP3), or Hyphantria cunea MNPV IAP1. In vAcΔp35-infected apoptotic Ld652Y cells, Apsup restricted apoptosis induction and prevented processing of endogenous Ld-Dronc. Conversely, upon RNA interference (RNAi)-mediated silencing of apsup, LdMNPV-infected Ld652Y cells, which typically support high-titer virus replication, underwent apoptosis, accompanied by the processing of endogenous Ld-Dronc. Furthermore, endogenous Ld-Dronc coimmunoprecipitated with transiently expressed Apsup, indicating that Apsup physically interacts with Ld-Dronc. Apsup prevented the apoptosis of Sf9 cells triggered by vAcΔp35 infection but did not inhibit apoptosis or activation of caspase-3-like protease in vAcΔp35-infected Drosophila melanogaster S2 cells. Apsup also inhibited the proteolytic processing of L. dispar effector caspase Ld-caspase-1 in the transient expression assay but did not physically interact with Ld-caspase-1. These results demonstrate that Apsup inhibits apoptosis in Ld652Y cells by preventing the proteolytic processing of Ld-Dronc. Together with our previous findings showing that Apsup prevents the processing of both overexpressed Ld-Dronc and Bombyx mori Dronc, these results also demonstrate that Apsup functions as an effective apoptotic suppressor in various lepidopteran, but not dipteran, insect cells.     

ORF390 of white spot syndrome virus genome is identified as a novel anti-apoptosis gene

Apoptosis serves as an important defense strategy employed by host cells against viral invasion. Many viruses contain the anti-apoptotic genes to block the defense-by-death response of host cells. In this study, we tried to identify the putative anti-apoptotic genes in white spot syndrome virus (WSSV) genome. We confirmed that actinomycin D could induce apoptosis of shrimp primary cells. However, the apoptosis triggered by actinomycin D was inhibited by WSSV infection. As mutants of Autographa californica nucleopolyhedrovirus (AcMNPV), AcMNPVDelta35k/pol+ lacks a functional P35 gene undergoing apoptosis and its infection could induce Sf9 cell apoptosis. To identify the putative apoptotic suppressor gene of WSSV, overlapping cosmid clones representing the entire WSSV genome were individually cotransfected along with genome DNA of AcMNPVDeltaP35k/pol+. Using this marker rescue assay, a WSSV DNA fragment that was able to rescue AcMNPVDeltaP35k/pol+ infection in Sf9 cells was isolated. By further sequence analysis and rescue assay, the ORF390 was identified as a novel anti-apoptotic gene. The ORF displays two putative caspase9 cleavage sites LLVETDGPS, VKLEHDGSK, and a caspase3 cleavage site EEDEVDGVP. The ORF was cloned into the pIE1 vector and then the recombinant vector was transfected into Sf9 cells. The Sf9 cells did not show obvious characteristics of apoptosis when infected with AcMNPVDeltaP35k/pol+. And the transient expression of ORF390 allowed AcMNPVDeltaP35k/pol+ replication in Sf9 cells and resulted in the formation of polyhedra successfully. The results indicate that function of ORF390 in WSSV is a kind of apoptotic suppressor like P35 in AcMNPV.     

Caspase Cleavage Sites in the Human Proteome: CaspDB,a Database of Predicted Substrates

Caspases are enzymes belonging to a conserved family of cysteine-dependent aspartic-specific proteases that are involved in vital cellular processes and play a prominent role in apoptosis and inflammation. Determining all relevant protein substrates of caspases remains a challenging task. Over 1500 caspase substrates have been discovered in the human proteome according to published data and new substrates are discovered on a daily basis. To aid the discovery process we developed a caspase cleavage prediction method using the recently published curated MerCASBA database of experimentally determined caspase substrates and a Random Forest classification method. On both internal and external test sets, the ranking of predicted cleavage positions is superior to all previously developed prediction methods. The in silico predicted caspase cleavage positions in human proteins are available from a relational database: CaspDB. Our database provides information about potential cleavage sites in a verified set of all human proteins collected in Uniprot and their orthologs, allowing for tracing of cleavage motif conservation. It also provides information about the positions of disease-annotated single nucleotide polymorphisms, and posttranslational modifications that may modulate the caspase cleaving efficiency.     

Caspase cleavage of the amyloid precursor protein modulates amyloid beta-protein toxicity

The amyloid beta-protein precursor (APP) is proteolytically cleaved to generate the amyloid beta-protein (Abeta), the principal constituent of senile plaques found in Alzheimer's disease (AD). In addition, Abeta in its oligomeric and fibrillar forms have been hypothesized to induce neuronal toxicity. We and others have previously shown that APP can be cleaved by caspases at the C-terminus to generate a potentially cytotoxic peptide termed C31. Furthermore, this cleavage event and caspase activation were increased in the brains of AD, but not control, cases. In this study, we show that in cultured cells, Abeta induces caspase cleavage of APP in the C-terminus and that the subsequent generation of C31 contributes to the apoptotic cell death associated with Abeta. Interestingly, both Abeta toxicity and C31 pathway are dependent on the presence of APP. Both APP-dependent Abeta toxicity and C31-induced apoptotic cell death involve apical or initiator caspases-8 and -9. Our results suggest that Abeta-mediated toxicity initiates a cascade of events that includes caspase activation and APP cleavage. These findings link C31 generation and its potential cell death activity to Abeta cytotoxicity, the leading mechanism proposed for neuronal death in AD.     

Caspase 8 inhibits programmed necrosis by processing CYLD

Caspase 8 initiates apoptosis downstream of TNF death receptors by undergoing autocleavage and processing the executioner caspase 3 (ref. 1). However, the dominant function of caspase 8 is to transmit a pro-survival signal that suppresses programmed necrosis (or necroptosis) mediated by RIPK1 and RIPK3 (refs 2-6) during embryogenesis and haematopoiesis(7-9). Suppression of necrotic cell death by caspase 8 requires its catalytic activity but not the autocleavage essential for apoptosis(10); however, the key substrate processed by caspase 8 to block necrosis has been elusive. A key substrate must meet three criteria: it must be essential for programmed necrosis; it must be cleaved by caspase 8 in situations where caspase 8 is blocking necrosis; and mutation of the caspase 8 processing site on the substrate should convert a pro-survival response to necrotic death without the need for caspase 8 inhibition. We now identify CYLD as a substrate for caspase 8 that satisfies these criteria. Following TNF stimulation, caspase 8 cleaves CYLD to generate a survival signal. In contrast, loss of caspase 8 prevented CYLD degradation, resulting in necrotic death. A CYLD substitution mutation at Asp 215 that cannot be cleaved by caspase 8 switches cell survival to necrotic cell death in response to TNF.     

Antioxidants prevent UV-induced apoptosis by inhibiting mitochondrial cytochrome c release and caspase activation in Spodoptera frugiperda (Sf9) cells

Oxidative stress has been shown to be associated with apoptosis (programmed cell death) in a number of cell systems. We earlier reported in vitro cultured Spodoptera frugiperda (Sf9) cells as a model system to study oxidative stress induced apoptosis (J Biosciences 24 (1999) 13) and the inhibition of UV-induced apoptosis by the baculovirus antiapoptotic p35 protein that acts as a sink to sequester reactive oxygen species (Proc Natl Acad Sci USA 96 (1999) 4838). We now show that UV-induced apoptosis in Sf9 cells, is preceded by the release of mitochondrial cytochrome c into the cytosol and consequent activation of Sf-caspase-1. The inhibitory effect of different antioxidants including scavengers of oxygen radicals such as butylated hydroxyanisole (BHA), alpha tocopherol acetate, benzoate and reduced-glutathione (GSH) on ultra violet B (UVB)-induced apoptosis in cultured Sf9 cells was assessed. Both, cytochrome c release as well as Sf-caspase-1 activation was inhibited by pre-treatment with antioxidants such as BHA and alpha tocopherol acetate, suggesting that these antioxidants inhibit apoptosis by acting quite upstream in the apoptosis cascade at the mitochondrial level, as well as downstream at the caspase level.     

Caspase 的活化机制

Caspase是一类与凋亡密切相关的蛋白水解酶家族,以Caspase前体酶原的形式存在大多后生动物的细胞中。Caspase在凋亡信号的作用下首先激活启动型Caspase引发Caspase级联反应,然后通过活化的执行型Caspase裂解特异性底物导致细胞凋亡。Caspase的活化是导致细胞凋亡的中心环节,位于Caspase级联反应上游的启动型Caspase的和下游的执行型Caspase有着明显不同的活化机制。     

Caspase Activation and Specific Cleavage of Substrates after Coxsackievirus B3-Induced Cytopathic Effect in HeLa Cells

Coxsackievirus B3 (CVB3), an enterovirus in the family Picornaviridae, induces cytopathic changes in cell culture systems and directly injures multiple susceptible organs and tissues in vivo, including the myocardium, early after infection. Biochemical analysis of the cell death pathway in CVB3-infected HeLa cells demonstrated that the 32-kDa proform of caspase 3 is cleaved subsequent to the degenerative morphological changes seen in infected HeLa cells. Caspase activation assays confirm that the cleaved caspase 3 is proteolytically active. The caspase 3 substrates poly(ADP-ribose) polymerase, a DNA repair enzyme, and DNA fragmentation factor, a cytoplasmic inhibitor of an endonuclease responsible for DNA fragmentation, were degraded at 9 h following infection, yielding their characteristic cleavage fragments. Inhibition of caspase activation by benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (ZVAD.fmk) did not inhibit the virus-induced cytopathic effect, while inhibition of caspase activation by ZVAD.fmk in control apoptotic cells induced by treatment with the porphyrin photosensitizer benzoporphyrin derivative monoacid ring A and visible light inhibited the apoptotic phenotype. Caspase activation and cleavage of substrates may not be responsible for the characteristic cytopathic effect produced by picornavirus infection yet may be related to late-stage alterations of cellular homeostatic processes and structural integrity.     

Caspase 7 can cleave tumor necrosis factor receptor-I (p60) at a non-consensus motif, in vitro.

Ligand binding to tumor necrosis factor receptor-I (TNFRI) can promote cell survival or activate the apoptotic caspase cascade. Cytoplasmic interaction of TNFRI with TRAF2 and RIP allows for the activation of JNK and NFkappaB pathways. Alternatively, a carboxy terminal death domain protein interaction motif can recruit TRADD, which then recruits FADD/MORT1, and finally procaspase 8. Aggregation of these components form a death inducing signaling complex, leading to the cleavage and activation of caspase 8. We have found that during apoptosis human TNFRI protein is lost in a caspase-dependent manner. The cytoplasmic tail of human TNFRI was found to be susceptible to caspase cleavage but not by caspase 8. Instead, the downstream executioner caspase 7 was the only caspase capable of cleaving TNFRI, in vitro. Identification and characterization of the cleavage site revealed a derivative of the classic EXD motif that incorporates a glutamate (E) in the P1 position. Using several criteria to establish that caspase activity was responsible for cleavage at this site, we confirmed that caspase 7 can cleave at a GELE motif. Mutation of the cleavage site prevented the apoptosis-associated cleavage of TNFRI. This ability of caspase 7 to cleave at a non-EXD or -DXXD motif suggests that the specificity of caspases may be broader than is currently held.     

A COMPREHENSIVE STUDY ON APOPTOSIS INDUCTION BY AZADIRACHTIN IN Spodoptera frugiperda CULTURED CELL LINE Sf9

The induction of apoptosis by azadirachtin, a well‐known botanical tetranortriterpenoid isolated from the neem tree (Azadirachta indica A. Juss) and other members of the Meliaceae, was investigated in Spodoptera frugiperda cultured cell line (Sf9). Morphological changes in Sf9 cells treated by various concentrations of azadirachtin were observed at different times under light microscopy. Morphological and biochemical analysis indicated that Sf9 cells treated by 1.5 μg/mL azadirachtin showed typical morphological changes, which were indicative of apoptosis and a clear DNA ladder. The flow cytometry analysis showed the apoptosis rate reached a maximum value of 32.66% at 24 h with 1.5 μg/mL azadirachtin in Sf9 cells. The inhibition of Sf9 cell proliferation suggested that the effect of azadirachtin was dose dependent and the EC₅₀ at 48 and 72 h was 2.727 × 10⁻⁶ and 6.348 × 10⁻⁹ μg/mL, respectively. The treatment of azadirachtin in Sf9 cells could significantly increase the activity of Sf caspase‐1, but showed no effect on the activity of Topo I, suggesting that the apoptosis induced by azadirachtinin Sf9 cells is through caspase‐dependent pathway. These results provided not only a series of morphological, biochemical, and toxicological comprehensive evidences for induction of apoptosis by azadirachtin, but also a reference model for screening insect cell apoptosis inducers from natural compounds.