C-terminal region of Bfl-1 induces cell death that accompanies caspase activation when fused with GFP

Previously, we reported that anti-apoptotic Bfl-1 is converted to a pro-apoptotic protein following fusion at its N-terminus with green fluorescent protein (GFP) (GFP-Bfl-1). In this study, we performed a Bfl-1 deletion study in order to elucidate the underlying mechanism of GFP-Bfl-1-induced cell death. We found that the Bcl-2 homology (BH) domains in Bfl-1 are dispensable with respect to cell death and that GFP fusion with the 29 amino acids of the C-terminal region of Bfl-1 (GFP-BC) is sufficient to induce cell death. Moreover, when BC was fused with other tagging partners like GST or MBP, little cell death was observed, implying that the GFP region is as important as the BC region for GFP-BC-induced cell death. Further deletion analysis defined a region of GFP as a determinant of GFP-BC-induced cell death. Confocal microscopic analysis showed that GFP-chimeras containing the BC region of Bfl-1 are located mainly in mitochondria. The GFP-BC-induced cell death accompanied cellular caspase activation, and treatment with the pan-caspase inhibitor, Boc-D-FMK, partially inhibited GFP-BC-induced cell death. However, the over-expression of anti-apoptotic molecules, such as Bcl-x(L) and CrmA, did not block GFP-BC-induced cell death. In summary, GFP-BC induces cell death with caspase activation through mitochondria dependent process.     

CD7 delivers a pro-apoptotic signal during galectin-1-induced T cell death

Galectin-1, an endogenous lectin expressed in lymphoid organs and immune-privileged sites, induces death of human and murine thymocytes and T cells. Galectin-1 binds to several glycoproteins on the T cell surface, including CD7. However, the T cell surface glycoprotein receptors responsible for delivering the galectin-1 death signal have not been identified. We show that CD7 is required for galectin-1-mediated death. This demonstrates a novel function for CD7 as a death trigger and identifies galectin-1/CD7 as a new biologic death signaling pair.     

The future of GI and liver research: editorial perspectives. III. JNK/AP-1 regulation of hepatocyte death

Activation of the JNK/activator protein-1 (AP-1)-signaling pathway is a common mediator of hepatocyte death from a variety of stimuli. Although the mechanism by which JNK or AP-1 promotes death is unknown, it results when activation of this signaling pathway is unusually prolonged. Although JNK/AP-1 mediates TNF-induced cell death at or above the level of the mitochondria, the ability of JNK/AP-1 to promote death from necrosis as well as apoptosis suggests that JNK/AP-1 may induce death by several mechanisms. Recognition of JNK/AP-1 signaling as a critical promoter of hepatocyte death raises the possibility that the therapeutic manipulation of this pathway may be effective in the treatment of human liver disease.     

β-Lapachone-induced reactive oxygen species (ROS) generation mediates autophagic cell death in glioma U87 MG cells

Autophagy is mainly responsible for the degradation of long-lived proteins and subcellular organelles. Autophagy is responsible for the non-apoptotic cell death, and plays a crucial role in regulating cellular functions. β-Lapachone is a quinone-containing compound originally obtained from the lapacho tree in South America. Here, we show that β-lapachone induces death in U87 MG cells, which is not inhibited by blockers of pan-caspase or necrosis. β-Lapachone-induced cell death gradually increased in a time-dependent manner in U87 MG cells, which were partly prevented by pretreatment of a specific inhibitor of NQO1 (dicoumarol). These results suggested that β-lapachone-induced cell death was mediated by NQO1-independent as well as NQO1-dependent cell death pathways. During progression of β-lapachone-induced cell death, translocation and processing of LC3 as well as an increase in acidic vesicular organelles, as assessed by acridine orange staining, were observed. Furthermore, β-lapachone-induced cell death was inhibited by either a knockdown of beclin-1/Atg-6 or Atg-7 gene expression or by autophagy inhibitors (3-methyl adenine or bafilomycin A1). Reactive oxygen species (ROS) were involved in β-lapachone-induced autophagic cell death of U87 MG glioma cells, because β-lapachone induced ROS production and antioxidant N-acetylcysteine (NAC) decreased autophagic cell death. Our results collectively demonstrate that ROS mediate β-lapachone-induced autophagic cell death in U87 MG glioma cells.     

Viral infection and the evolution of caspase 8-regulated apoptotic and necrotic death pathways

Pathogens specifically target both the caspase 8-dependent apoptotic cell death pathway and the necrotic cell death pathway that is dependent on receptor-interacting protein 1 (RIP1; also known as RIPK1) and RIP3 (also known as RIPK3). The fundamental co-regulation of these two cell death pathways emerged when the midgestational death of mice deficient in FAS-associated death domain protein (FADD) or caspase 8 was reversed by elimination of RIP1 or RIP3, indicating a far more entwined relationship than previously appreciated. Thus, mammals require caspase 8 activity during embryogenesis to suppress the kinases RIP1 and RIP3 as part of the dialogue between two distinct cell death processes that together fulfil reinforcing roles in the host defence against intracellular pathogens such as herpesviruses.     

Protective role of Cop in Rip2/caspase-1/caspase-4-mediated HeLa cell death

CARD only protein (Cop) was recently identified as a protein with significant homology with the CARD of caspase-1. We have conducted functional studies on Cop and report on its role as an inhibitor of cell death in a broad range of cell death paradigms. A notable exception in the ability of Cop to inhibit cell death pertains to its inability to inhibit ER stress-mediated cell death. Furthermore, in addition to the known interaction of Cop and caspase-1, we demonstrated a novel interaction of Cop with caspase-4. We propose that Cop's action to prevent TNF-alpha-induced cell death may operate independently of the mitochondrial death pathway. Furthermore, Cop overexpression inhibits Bid cleavage. In summary, Cop inhibition of cell death, at least to a certain extent, results from its interference with the activation of caspase-1 and caspase-4. Understanding the mechanistic details modulating caspase cell death pathways should provide important information for the development of therapies for diseases featuring aberrant caspase activation. Cop, as an inhibitor of an important apical caspase cell death axis, may provide a tool for modulating pathological cell death.     

Deletion of a chaperonin 60 beta gene leads to cell death in the Arabidopsis lesion initiation 1 mutant

Lesion mimic mutants develop spontaneous cell death without pathogen attack. Some of the genes defined by these mutations may function as regulators of cell death, whereas others may perturb cellular metabolism in a way that leads to cell death. To understand the molecular mechanism of cell death in lesion mimic mutants, we isolated a lesion initiation 1 (len1) mutant by a T-DNA tagging method. The len1 mutant develops lesions on its leaves and expresses systemic acquired resistance (SAR). LEN1 was identified to encode a chloroplast chaperonin 60 beta (Cpn60 beta), a homologue of bacterial GroEL. The recombinant LEN1 had molecular chaperone activity for suppressing protein aggregation in vitro. Moreover, len1 plants develop accelerated cell death to heat shock stress in comparison with wild-type plants. The chlorophyll a/b binding protein (CAB) was present in len1 plants at a lower level than in the wild-type plants. These results indicate that LEN1 functions as a molecular chaperone in chloroplasts and its deletion leads to cell death in Arabidopsis.     

The Drosophila caspase DRONC cleaves following glutamate or aspartate and is regulated by DIAP1, HID, and GRIM

The caspase family of cysteine proteases plays important roles in bringing about apoptotic cell death. All caspases studied to date cleave substrates COOH-terminal to an aspartate. Here we show that the Drosophila caspase DRONC cleaves COOH-terminal to glutamate as well as aspartate. DRONC autoprocesses itself following a glutamate residue, but processes a second caspase, drICE, following an aspartate. DRONC prefers tetrapeptide substrates in which aliphatic amino acids are present at the P2 position, and the P1 residue can be either aspartate or glutamate. Expression of a dominant negative form of DRONC blocks cell death induced by the Drosophila cell death activators reaper, hid, and grim, and DRONC overexpression in flies promotes cell death. Furthermore, the Drosophila cell death inhibitor DIAP1 inhibits DRONC activity in yeast, and DIAP1's ability to inhibit DRONC-dependent yeast cell death is suppressed by HID and GRIM. These observations suggest that DRONC acts to promote cell death. However, DRONC activity is not suppressed by the caspase inhibitor and cell death suppressor baculovirus p35. We discuss possible models for DRONC function as a cell death inhibitor.     

TAK1 is a master regulator of epidermal homeostasis involving skin inflammation and apoptosis

Transforming growth factor beta-activated kinase 1 (TAK1) functions downstream of inflammatory cytokines to activate c-Jun N-terminal kinase (JNK) as well as NF-kappaB in several cell types. However, the functional role of TAK1 in an in vivo setting has not been determined. Here we have demonstrated that TAK1 is the major regulator of skin inflammation as well as keratinocyte death in vivo. Epidermal-specific deletion of TAK1 causes a severe inflammatory skin condition by postnatal day 6-8. The mutant skin also exhibits massive keratinocyte death. Analysis of keratinocytes isolated from the mutant skin revealed that TAK1 deficiency results in a striking increase in apoptosis in response to tumor necrosis factor (TNF). TAK1-deficient keratinocytes cannot activate NF-kappaB or JNK upon TNF treatment. These results suggest that TNF induces TAK1-deficient keratinocyte death because of the lack of NF-kappaB (and possibly JNK)-mediated cell survival signaling. Finally, we have shown that deletion of the TNF receptor can largely rescue keratinocyte death as well as inflammatory skin condition in epidermal-specific TAK1-deficient mice. Our results demonstrate that TAK1 is a master regulator of TNF signaling in skin and regulates skin inflammation and keratinocyte death.     

Bax inhibitor-1: a highly conserved endoplasmic reticulum-resident cell death suppressor

In spite of fundamental differences between plant and animal cells, it is remarkable that some cell death regulators that were identified to control cell death in metazoans can also function in plants. The fact that most of these proteins do not have structural homologs in plant genomes suggests that they may be targeting a highly conserved 'core' mechanism with conserved functions that is present in all eukaryotes. The ubiquitous Bax inhibitor-1 (BI-1) is a common cell death suppressor in eukaryotes that has provided a potential portal to this cell death core. In this review, we will update the current status of our understanding on the function and activities of this intriguing protein. Genetic, molecular and biochemical studies have so far suggested a consistent view that BI-1 is an endoplasmic reticulum (ER)-resident transmembrane protein that can interact with multiple partners to alter intracellular Ca(2+) flux control and lipid dynamics. Functionally, the level of BI-1 protein has been hypothesized to have the role of a rheostat to regulate the threshold of ER-stress inducible cell death. Further, delineation of the cell death suppression mechanism by BI-1 should shed light on an ancient cell death core-control pathway in eukaryotes, as well as novel ways to improve stress tolerance.     

Arabidopsis Bax inhibitor-1 functions as an attenuator of biotic and abiotic types of cell death

Programmed cell death (PCD) is a common process in eukaryotes during development and in response to pathogens and stress signals. Bax inihibitor-1 (BI-1) is proposed to be a cell death suppressor that is conserved in both animals and plants, but the physiological importance of BI-1 and the impact of its loss of function in plants are still unclear. In this study, we identified and characterized two independent Arabidopsis mutants with a T-DNA insertion in the AtBI1 gene. The phenotype of atbi1-1 and atbi1-2, with a C-terminal missense mutation and a gene knockout, respectively, was indistinguishable from wild-type plants under normal growth conditions. However, these two mutants exhibit accelerated progression of cell death upon infiltration of leaf tissues with a PCD-inducing fungal toxin fumonisin B1 (FB1) and increased sensitivity to heat shock-induced cell death. Under these conditions, expression of AtBI1 mRNA was up-regulated in wild-type leaves prior to the activation of cell death, suggesting that increase of AtBI1 expression is important for basal suppression of cell death progression. Over-expression of AtBI1 transgene in the two homozygous mutant backgrounds rescued the accelerated cell death phenotypes. Together, our results provide direct genetic evidence for a role of BI-1 as an attenuator for cell death progression triggered by both biotic and abiotic types of cell death signals in Arabidopsis.     

c-di-GMP induction of Dictyostelium cell death requires the polyketide DIF-1

Cell death in the model organism Dictyostelium, as studied in monolayers in vitro, can be induced by the polyketide DIF-1 or by the cyclical dinucleotide c-di-GMP. c-di-GMP, a universal bacterial second messenger, can trigger innate immunity in bacterially infected animal cells and is involved in developmental cell death in Dictyostelium. We show here that c-di-GMP was not sufficient to induce cell death in Dictyostelium cell monolayers. Unexpectedly, it also required the DIF-1 polyketide. The latter could be exogenous, as revealed by a telling synergy between c-di-GMP and DIF-1. The required DIF-1 polyketide could also be endogenous, as shown by the inability of c-di-GMP to induce cell death in Dictyostelium HMX44A cells and DH1 cells upon pharmacological or genetic inhibition of DIF-1 biosynthesis. In these cases, c-di-GMP–induced cell death was rescued by complementation with exogenous DIF-1. Taken together, these results demonstrated that c-di-GMP could trigger cell death in Dictyostelium only in the presence of the DIF-1 polyketide or its metabolites. This identified another element of control to this cell death and perhaps also to c-di-GMP effects in other situations and organisms.     

Molecular genetics of cell death in the nematode Caenorhabditis elegans.

In C. elegans, cell death can be readily studied at the cellular, genetic, and molecular levels. Two types of death have been characterized in this nematode: (1) programmed cell death, which occurs as a normal component in development; and (2) pathological cell death, which occurs aberrantly as a consequence of mutation. Analysis of mutations that disrupt programmed cell death in various ways has defined a genetic pathway for programmed cell death which includes genes that perform such functions as the determination of which cells die, the execution of cell death, the engulfment of cell corpses, and the digestion of DNA from dead cells. Molecular analysis is providing insight into the nature of the molecules that function in these aspects of programmed cell death. Characterization of some genes that mutate to induce abnormal cell death has defined a novel gene family called degenerins that encode putative membrane proteins. Dominant alleles of at least two degenerin genes, mec-4 and deg-1, can cause cellular swelling and late onset neurodegeneration of specific groups of cells.     

The tumor suppressor RASSF1A and MAP-1 link death receptor signaling to Bax conformational change and cell death

Tumor cells typically resist programmed cell death (apoptosis) induced by death receptors. Activated death receptors evoke Bax conformational change, cytochrome c release, and cell death. We report that the tumor suppressor gene RASSF1A is required for death receptor-induced Bax conformational change and apoptosis. TNFalpha or TRAIL stimulation induced recruitment of RASSF1A and MAP-1 to receptor complexes and promoted complex formation between RASSF1A and the BH3-like protein MAP-1. Normally, MAP-1 is inhibited by an intramolecular interaction. RASSF1A/MAP-1 binding relieved this inhibitory interaction, resulting in MAP-1 association with Bax. Deletion of the RASSF1A gene or short hairpin silencing of either RASSF1A or MAP-1 expression blocked MAP-1/Bax interaction, Bax conformational change and mitochondrial membrane insertion, cytochrome c release, and apoptosis in response to death receptors. Our findings identify RASSF1A and MAP-1 as important components between death receptors and the apoptotic machinery and reveal a potential link between tumor suppression and death receptor signaling.     

HSpin1, a transmembrane protein interacting with Bcl-2/Bcl-xL,induces a caspase-independent autophagic cell death

The Drosophila spinster (spin) gene product is required for programmed cell death in the nervous and reproductive systems. We have identified a human homologue of the Drosophila spin gene product (HSpin1). HSpin1 bound to Bcl-2 and apoptosis regulator Bcl-X (Bcl-xL), but not to proapoptotic members such as Bcl-2-associated X protein and Bcl-2 homologous antagonist killer, in cells treated with TNF-alpha. Exogenous expression of HSpin1 resulted in the cell death without inducing a release of cytochrome c from mitochondria. Overexpression of Bcl-xL inhibited the HSpin1-induced cell death. Interestingly, a necrosis inhibitor, pyrrolidine dithiocarbomate, but not the pancaspase inhibitors, carbobenzoxy-VAD-fluoromethyl ketone and p35, blocked the HSpin1-induced cell death. HSpin1-induced cell death increases autophagic vacuole and mature form of cathepsin D, suggesting a novel caspase-independent cell death, which is link to autophagy.     

The plant gene CCD1 selectively blocks cell death during the hypersensitive response to Cauliflower mosaic virus infection

The P6 protein of Cauliflower mosaic virus (CaMV) W260 elicits a hypersensitive response (HR) on inoculated leaves of Nicotiana edwardsonii. This defense response, common to many plant pathogens, has two key characteristics, cell death within the initially infected tissues and restriction of the pathogen to this area. We present evidence that a plant gene designated CCD1, originally identified in N. bigelovii, can selectively block the cell death pathway during HR, whereas the resistance pathway against W260 remains intact. Suppression of cell death was evident not only macroscopically but also microscopically. The suppression of HR-mediated cell death was specific to CaMV, as Tobacco mosaic virus was able to elicit HR in the plants that contained CCD1. CCD1 also blocks the development of a systemic cell death symptom induced specifically by the P6 protein of W260 in N. clevelandii. Introgression of CCD1 from N. bigelovii into N. clevelandii blocked the development of systemic cell death in response to W260 infection but could not prevent systemic cell death induced by Tomato bushy stunt virus. Thus, CCD1 blocks both local and systemic cell death induced by P6 of W260 but does not act as a general suppressor of cell death induced by other plant viruses. Furthermore, experiments with CCD1 provide further evidence that cell death could be uncoupled from resistance in the HR of Nicotiana edwardsonii to CaMV W260.     

Caspase-1: is IL-1 just the tip of the ICEberg?

Caspase-1, formerly known as interleukin (IL)-1-converting enzyme is best established as the protease responsible for the processing of the key pro-inflammatory cytokine IL-1β from an inactive precursor to an active, secreted molecule. Thus, caspase-1 is regarded as a key mediator of inflammatory processes, and has become synonymous with inflammation. In addition to the processing of IL-1β, caspase-1 also executes a rapid programme of cell death, termed pyroptosis, in macrophages in response to intracellular bacteria. Pyroptosis is also regarded as a host response to remove the niche of the bacteria and to hasten their demise. These processes are generally accepted as the main roles of caspase-1. However, there is also a wealth of literature supporting a direct role for caspase-1 in non-infectious cell death processes. This is true in mammals, but also in non-mammalian vertebrates where caspase-1-dependent processing of IL-1β is absent because of the lack of appropriate caspase-1 cleavage sites. This literature is most prevalent in the brain where caspase-1 may directly regulate neuronal cell death in response to diverse insults. We attempt here to summarise the evidence for caspase-1 as a cell death enzyme and propose that, in addition to the processing of IL-1β, caspase-1 has an important and a conserved role as a cell death protease.