Granzyme A cleaves a mitochondrial complex I protein to initiate caspase-independent cell death

The killer lymphocyte protease granzyme A (GzmA) triggers caspase-independent target cell death with morphological features of apoptosis. We previously showed that GzmA acts directly on mitochondria to generate reactive oxygen species (ROS) and disrupt the transmembrane potential (DeltaPsi(m)) but does not permeabilize the mitochondrial outer membrane. Mitochondrial damage is critical to GzmA-induced cell death since cells treated with superoxide scavengers are resistant to GzmA. Here we find that GzmA accesses the mitochondrial matrix to cleave the complex I protein NDUFS3, an iron-sulfur subunit of the NADH:ubiquinone oxidoreductase complex I, after Lys56 to interfere with NADH oxidation and generate superoxide anions. Target cells expressing a cleavage site mutant of NDUFS3 are resistant to GzmA-mediated cell death but remain sensitive to GzmB.     

Granzyme a, a stealth killer in the mitochondrion

The induction of caspase-independent cell death by killer lymphocytes involves the serine protease granzyme A (GzmA). In this issue, Martinvalet et al. (2008) show that GzmA penetrates the mitochondrial matrix without perturbing normal mitochondrial functions. In the mitochondrial matrix, GzmA cleaves NDUFS3 (a component of the electron transport chain) leading to production of reactive oxygen species and ultimately to cell death.     

Granzyme K cleaves the nucleosome assembly protein SET to induce single-stranded DNA nicks of target cells

Although granzymes (Gzms) A- and B-induced cell death pathways have been defined, little is known about how other orphan Gzms function in CTL-mediated cytotoxicity. GzmK and A are tryptases among all the Gzms of humans and they are closely linked on the same chromosome. In this study, we showed that GzmK can be efficiently delivered into target cells with a cationic lipid protein transfection reagent Pro-Ject. We found human GzmK triggers rapid cell death independently of caspase activation. The features of death are characterized by rapid externalization of phosphatidylserine, nuclear morphological changes and single-stranded DNA nicks. GzmK hydrolyzes the nucleosome assembly protein SET in its recombinant and native forms or in intact cells. Cleavage of SET by GzmK abrogates its nucleosome assembly activity. After GzmK loading, SET and DNase NM23H1 rapidly translocate into the nucleus and SET is cleaved, where the nuclease activity of NM23H1 is activated to nick chromosomal DNA.     

Biological activities of granzyme K are conserved in the mouse and account for residual Z-Lys-SBzl activity in granzyme A-deficient mice.

Tryptase-like activities of T and NK cells contribute to the induction of target cell apoptosis, but only granzyme A (GzmA) has been shown to exhibit Z-Lys-SBzl esterase activity in murine T cells. GzmA-deficient mice exhibit residual Z-Lys-SBzl hydrolyzing activity and almost normal levels of lymphocyte-mediated cytotoxicity. Here we report the cloning and biochemical characterization of recombinant mouse granzyme K (GzmK). The purified murine protein shows Z-Lys-SBzl hydrolyzing activity and is inhibited by bikunin, the light chain of inter-alpha-trypsin inhibitor, like the human homolog. We conclude that GzmK expressed by GzmA-deficient T cells accounts for the remaining Z-Lys-SBzl activity. Functional similarities between GzmA and GzmK may explain the subtle immunological deficits observed in GzmA-deficient mice.     

Tumor suppressor NM23-H1 is a granzyme A-activated DNase during CTL-mediated apoptosis,and the nucleosome assembly protein SET is its inhibitor

Granzyme A (GzmA) induces a caspase-independent cell death pathway characterized by single-stranded DNA nicks and other features of apoptosis. A GzmA-activated DNase (GAAD) is in an ER associated complex containing pp32 and the GzmA substrates SET, HMG-2, and Ape1. We show that GAAD is NM23-H1, a nucleoside diphosphate kinase implicated in suppression of tumor metastasis, and its specific inhibitor (IGAAD) is SET. NM23-H1 binds to SET and is released from inhibition by GzmA cleavage of SET. After GzmA loading or CTL attack, SET and NM23-H1 translocate to the nucleus and SET is degraded, allowing NM23-H1 to nick chromosomal DNA. GzmA-treated cells with silenced NM23-H1 expression are resistant to GzmA-mediated DNA damage and cytolysis, while cells overexpressing NM23-H1 are more sensitive.     

Cooperation of phosphatidylcholine-specific phospholipase C and basic fibroblast growth factor in the neural differentiation of mesenchymal stem cells in vitro

Previously, we found that suppressing phosphatidylcholine-specific phospholipase C could induce neuronal differentiation of rat mesenchymal stem cells in the absence of serum and fibroblast growth factor. It is well known that basic fibroblast growth factor plays an important role in mesenchymal stem cell neuronal differentiation. In this study, our purpose was to understand the cooperation of phosphatidylcholine-specific phospholipase C and basic fibroblast growth factor in controlling mesenchymal stem cell neuronal differentiation. Our results showed that suppressing phosphatidylcholine-specific phospholipase C in the presence of basic fibroblast growth factor could induce cell neuronal differentiation and the viability of the differentiated cells was obviously increased. Furthermore, we found that the resting membrane potential of the differentiated cells gradually decreased, but the mitochondrial membrane potential rose with increasing treatment time and these characteristics were similar to cultured neurons from mouse embryo forebrains. To determine the possible mechanism by which this combination controls cell neuronal differentiation, we measured changes in the mitochondrial membrane potential and in the levels of reactive oxygen species. The results showed that both the mitochondrial membrane potential and reactive oxygen species levels decreased when basic fibroblast growth factor was added. The data suggested that lower phosphatidylcholine-specific phospholipase C activity was required for mesenchymal stem cell neuronal differentiation and basic fibroblast growth factor was necessary for maintaining the neuronal differentiation state. Moreover, basic fibroblast growth factor could contribute to rescuing the differentiated cells from death through decreasing overly high mitochondrial membrane potentials and reactive oxygen species levels.     

Granzyme A, which causes single-stranded DNA damage, targets the double-strand break repair protein Ku70

Granzyme A (GzmA) induces caspase-independent cell death with morphological features of apoptosis. Here, we show that GzmA at nanomolar concentrations cleaves Ku70, a key double-strand break repair (DSBR) protein, in target cells. Ku70 is cut after Arg(301), disrupting Ku complex binding to DNA. Cleaving Ku70 facilitates GzmA-mediated cell death, as silencing Ku70 by RNA interference increases DNA damage and cell death by GzmB cluster-deficient cytotoxic T lymphocytes or by GzmA and perforin, whereas Ku70 overexpression has the opposite effect. Ku70 has two known antiapoptotic effects-facilitating DSBR and sequestering bax to prevent its translocation to mitochondria. However, GzmA triggers single-stranded, not double-stranded, DNA damage, and GzmA-induced cell death does not involve bax. Therefore, Ku70 has other antiapoptotic functions in GzmA-induced cell death, which are blocked when GzmA proteolyses Ku70.     

Cardiolipin-mediated temporal response to hydroquinone toxicity in human retinal pigmented epithelial cell line

Hydroquinone, a potent toxic agent of cigarette smoke, damages retinal pigmented epithelial cells by triggering oxidative stress and mitochondrial dysfunction, two events causally related to the development and progression of retinal diseases. The inner mitochondrial membrane is enriched in cardiolipin, a phospholipid susceptible of oxidative modifications which determine cell-fate decision. Using ARPE-19 cell line as a model of retinal pigmented epithelium, we analyzed the potential involvement of cardiolipin in hydroquinone toxicity. Hydroquinone exposure caused an early concentration-dependent increase in mitochondrial reactive oxygen species, decrease in mitochondrial membrane potential, and rise in the rate of oxygen consumption not accompanied by changes in ATP levels. Despite mitochondrial impairment, cell viability was preserved. Hydroquinone induced cardiolipin translocation to the outer mitochondrial membrane, and an increase in the colocalization of the autophagosome adapter protein LC3 with mitochondria, indicating the induction of protective mitophagy. A prolonged hydroquinone treatment induced pyroptotic cell death by cardiolipin-mediated caspase-1 and gasdermin-D activation. Cardiolipin-specific antioxidants counteracted hydroquinone effects pointing out that cardiolipin can act as a mitochondrial “eat-me signal” or as a pyroptotic cell death trigger. Our results indicate that cardiolipin may act as a timer for the mitophagy to pyroptosis switch and propose cardiolipin-targeting compounds as promising approaches for the treatment of oxidative stress-related retinal diseases.     

BNIP3 and genetic control of necrosis-like cell death through the mitochondrial permeability transition pore

Many apoptotic signaling pathways are directed to mitochondria, where they initiate the release of apoptogenic proteins and open the proposed mitochondrial permeability transition (PT) pore that ultimately results in the activation of the caspase proteases responsible for cell disassembly. BNIP3 (formerly NIP3) is a member of the Bcl-2 family that is expressed in mitochondria and induces apoptosis without a functional BH3 domain. We report that endogenous BNIP3 is loosely associated with mitochondrial membrane in normal tissue but fully integrates into the mitochondrial outer membrane with the N terminus in the cytoplasm and the C terminus in the membrane during induction of cell death. Surprisingly, BNIP3-mediated cell death is independent of Apaf-1, caspase activation, cytochrome c release, and nuclear translocation of apoptosis-inducing factor. However, cells transfected with BNIP3 exhibit early plasma membrane permeability, mitochondrial damage, extensive cytoplasmic vacuolation, and mitochondrial autophagy, yielding a morphotype that is typical of necrosis. These changes were accompanied by rapid and profound mitochondrial dysfunction characterized by opening of the mitochondrial PT pore, proton electrochemical gradient (Deltapsim) suppression, and increased reactive oxygen species production. The PT pore inhibitors cyclosporin A and bongkrekic acid blocked mitochondrial dysregulation and cell death. We propose that BNIP3 is a gene that mediates a necrosis-like cell death through PT pore opening and mitochondrial dysfunction.     

The H-ATP synthase: A gate to ROS-mediated cell death or cell survival

Cellular oxidative stress results from the increased generation of reactive oxygen species and/or the dysfunction of the antioxidant systems. Most intracellular reactive oxygen species derive from superoxide radical although the majority of the biological effects of reactive oxygen species are mediated by hydrogen peroxide. In this contribution we overview the major cellular sites of reactive oxygen species production, with special emphasis in the mitochondrial pathways. Reactive oxygen species regulate signaling pathways involved in promoting survival and cell death, proliferation, metabolic regulation, the activation of the antioxidant response, the control of iron metabolism and Ca^2 + signaling. The reversible oxidation of cysteines in transducers of reactive oxygen species is the primary mechanism of regulation of the activity of these proteins. Next, we present the mitochondrial H⁺-ATP synthase as a core hub in energy and cell death regulation, defining both the rate of energy metabolism and the reactive oxygen species-mediated cell death in response to chemotherapy. Two main mechanisms that affect the expression and activity of the H⁺-ATP synthase down-regulate oxidative phosphorylation in prevalent human carcinomas. In this context, we emphasize the prominent role played by the ATPase Inhibitory Factor 1 in human carcinogenesis as an inhibitor of the H⁺-ATP synthase activity and a mediator of cell survival. The ATPase Inhibitory Factor 1 promotes metabolic rewiring to an enhanced aerobic glycolysis and the subsequent production of mitochondrial reactive oxygen species. The generated reactive oxygen species are able to reprogram the nucleus to support tumor development by arresting cell death. Overall, we discuss the cross-talk between reactive oxygen species signaling and mitochondrial function that is crucial in determining the cellular fate. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.     

Yeast caspase 1 links messenger RNA stability to apoptosis in yeast

During the past years, yeasts have been successfully established as models to study the mechanisms of apoptotic regulation. We recently showed that mutations in the LSM4 gene, which is involved in messenger RNA decapping, lead to increased mRNA stability and apoptosis in yeast. Here, we show that mitochondrial function and YCA1, which encodes a budding yeast metacaspase, are necessary for apoptosis triggered by stabilization of mRNAs. Deletion of YCA1 in yeast cells mutated in the LSM4 gene prevents mitochondrial fragmentation and rapid cell death during chronological ageing of the culture, diminishes reactive oxygen species accumulation and DNA breakage, and increases resistance to H2O2 and acetic acid. mRNA levels in lsm4 mutants deleted for YCA1 are still increased, positioning the Yca1 budding yeast caspase as a downstream executor of cell death induced by mRNA perturbations. In addition, we show that mitochondrial function is necessary for fast death during chronological ageing, as well as in LSM4 mutated and wild-type cells.     

Silver Nanoparticle Exposure Induced Mitochondrial Stress,Caspase-3 Activation and Cell Death: Amelioration by Sodium Selenite

Silver nanoparticles (AgNP), one of the most commonly used engineered nanomaterial for biomedical and industrial applications, has shown a toxic potential to our ecosystems and humans. In this study, murine hippocampal neuronal HT22 cells were used to delineate subcellular responses and mechanisms to AgNP by assessing the response levels of caspase-3, mitochondrial oxygen consumption, reactive oxygen species (ROS), and mitochondrial membrane potential in addition to cell viability testing. Selenium, an essential trace element that has been known to carry protecting property from heavy metals, was tested for its ameliorating potential in the cells exposed to AgNP. Results showed that AgNP reduced cell viability. The toxicity was associated with mitochondrial membrane depolarization, increased accumulation of ROS, elevated mitochondrial oxygen consumption, and caspase-3 activation. Treatment with sodium selenite reduced cell death, stabilized mitochondrial membrane potential and oxygen consumption rate, and prevented accumulation of ROS and activation of caspase-3. It is concluded that AgNP induces mitochondrial stress and treatment with selenite is capable of preventing the adverse effects of AgNP on the mitochondria.     

Mitochondrial superoxide production contributes to vancomycin-induced renal tubular cell apoptosis

Vancomycin chloride (VCM), a glycopeptide antibiotic, is widely used for the therapy of infections caused by methicillin-resistant Staphylococcus aureus. However, nephrotoxicity is a major adverse effect in VCM therapy. In this study, we investigated the cellular mechanisms underlying VCM-induced renal tubular cell injury in cultured LLC-PK1 cells. VCM induced a concentration- and time-dependent cell injury in LLC-PK1 cells. VCM caused increases in the numbers of annexin V-positive/PI-negative cells and TUNEL-positive cells, indicating the involvement of apoptotic cell death in VCM-induced renal cell injury. The VCM-induced apoptosis was accompanied by the activation of caspase-9 and caspase-3/7 and reversed by inhibitors of these caspases. Moreover, VCM caused an increase in intracellular reactive oxygen species production and mitochondrial membrane depolarization, which were reversed by vitamin E. In addition, mitochondrial complex I activity was inhibited by VCM as well as by the complex I inhibitor rotenone, and rotenone mimicked the VCM-induced LLC-PK1 cell injury. These findings suggest that VCM causes apoptotic cell death in LLC-PK1 cells by enhancing mitochondrial superoxide production leading to mitochondrial membrane depolarization followed by the caspase activities. Moreover, mitochondrial complex I may play an important role in superoxide production and renal tubular cell apoptosis induced by VCM.     

Mitochondrial electron transport chain activity is not involved in ultraviolet A (UVA)-induced cell death

Ultraviolet A (UVA), the long wavelength part of the sun's ultraviolet radiation, elicits its harmful effects through production of reactive oxygen species. In this study, we have tested the hypothesis that the mitochondrial electron transport chain, the main source of reactive oxygen species in cells, importantly contributes to UVA-induced cell damage. Model cell lines completely lacking a mitochondrial electron transport chain (rho(0)-cells) were not protected against UVA-induced cell death. Also, primary human fibroblasts and keratinocytes with induced depletion of electron transport chain activity were not better protected against UVA-induced cell death. On the other hand, diphenyleneiodonium and resiniferatoxin, inhibitors of plasma membrane oxidases, protected primary human fibroblasts against UVA, as potently as the lipid peroxidation chain breaker Trolox. These data indicate that plasma membrane electron transport systems, but not the mitochondrial electron transport chain, play a major role in UVA-induced cell death.     

Reactive oxygen species are required for hyperoxia-induced Bax activation and cell death in alveolar epithelial cells

Exposure of animals to hyperoxia results in respiratory failure and death within 72 h. Histologic evaluation of the lungs of these animals demonstrates epithelial apoptosis and necrosis. Although the generation of reactive oxygen species (ROS) is widely thought to be responsible for the cell death observed following exposure to hyperoxia, it is not clear whether they act upstream of activation of the cell death pathway or whether they are generated as a result of mitochondrial membrane permeabilization and caspase activation. We hypothesized that the generation of ROS was required for hyperoxia-induced cell death upstream of Bax activation. In primary rat alveolar epithelial cells, we found that exposure to hyperoxia resulted in the generation of ROS that was completely prevented by the administration of the combined superoxide dismutase/catalase mimetic EUK-134 (Eukarion, Inc., Bedford, MA). Exposure to hyperoxia resulted in the activation of Bax at the mitochondrial membrane, cytochrome c release, and cell death. The administration of EUK-134 prevented Bax activation, cytochrome c release, and cell death. In a mouse lung epithelial cell line (MLE-12), the overexpression of Bcl-XL protected cells against hyperoxia by preventing the activation of Bax at the mitochondrial membrane. We conclude that exposure to hyperoxia results in Bax activation at the mitochondrial membrane and subsequent cytochrome c release. Bax activation at the mitochondrial membrane requires the generation of ROS and can be prevented by the overexpression of Bcl-XL.     

Increased mitochondrial respiration maintains the mitochondrial membrane potential and promotes survival of cerebellar neurons in an endogenous model of glutamate receptor activation

It is thought that the combination of extracellular glutamate accumulation and mitochondrial damage is involved in neuronal death associated with brain ischemia and hypoglycemia, and some neurodegenerative diseases such as Huntington's disease. However, the mechanism whereby those two factors interact together to trigger neurodegeneration in this and other neurodegenerative disorders is still elusive. Here, we have addressed this issue using a model of mild and sustained accumulation of extracellular glutamate in cerebellar cultured neurons, which are mostly glutamatergic and commonly used to study glutamate neurotoxicity. The resulting stimulation of glutamate receptors triggered a approximately 50% persistent increase in mitochondrial respiration that was associated with free radicals formation, and which was found to be necessary to prevent the collapse of the mitochondrial membrane potential (Deltapsim) and apoptotic cell death. In fact, hampering the glutamate-mediated increase in mitochondrial respiration with an inhibitor of the mitochondrial respiratory chain stopped neurons from producing free radicals, but led them to undergo rapid and profound Deltapsim collapse and apoptotic cell death. Thus, we suggest that the formation of reactive oxygen species by glutamate receptor activation is the unavoidable consequence of an increase in the mitochondrial respiration aimed to prevent Deltapsim collapse and neurodegeneration. These results may be relevant to understand the pathophysiology of those neurodegenerative diseases associated with both mitochondrial respiratory chain and glutamate transporter defects.     

Potentiation of Fas-mediated apoptosis by attenuated production of mitochondria-derived reactive oxygen species

The role of reactive oxygen species (ROS) production in death receptor-mediated apoptosis is ill-defined. Here, we show that ROS levels play a role in moderating Fas-dependent apoptosis. Treatment of Jurkat T cells with oligomycin (ATP-synthase inhibitor) or (mitochondrial uncoupler) and Fas-activating antibody (CH11) facilitated rapid cell death that was not associated with decreased ATP production or increased DEVDase activity and cytochrome c release. However, a decrease in cellular ROS production was associated with CH11 treatment, and combinations of CH11 with oligomycin or FCCP further inhibited cellular ROS production. Thus, decreased ROS production is correlated with enhanced cell death. A transition from state 3 to state 4 mitochondrial respiration accounted for the attenuated ROS production and membrane potential. Similar observations were demonstrated in isolated rat liver mitochondria. These data show that ROS production is important in receptor-mediated apoptosis, playing a pivotal role in cell survival.     

Apoptosis in neurodegenerative diseases: the role of mitochondria

Nerve cell death is the central feature of the human neurodegenerative diseases. It has long been thought that nerve cell death in these disorders occurs by way of necrosis, a process characterized by massive transmembrane ion currents, compromise of mitochondrial ATP production, and the formation of high levels of reactive oxygen species combining to induce rapid disruption of organelles, cell swelling, and plasma membrane rupture with a secondary inflammatory response. Nuclear DNA is relatively preserved. Recent evidence now indicates that the process of apoptosis rather than necrosis primarily contributes to nerve cell death in neurodegeneration. This has opened up new avenues for understanding the pathogenesis of neurodegeneration and may lead to new and more effective therapeutic approaches to these diseases.     

Granzyme A activates an endoplasmic reticulum-associated caspase-independent nuclease to induce single-stranded DNA nicks

The cytotoxic T lymphocyte protease granzyme A (GzmA) initiates a novel caspase-independent cell death pathway characterized by single-stranded DNA nicking. The previously identified GzmA substrate SET is in a multimeric 270-420-kDa endoplasmic reticulum-associated complex that also contains the tumor suppressor protein pp32. GzmA cleaved the nucleosome assembly protein SET after Lys(176) and disrupted its nucleosome assembly activity. The purified SET complex required only GzmA to reconstitute single-stranded DNA nicking in isolated nuclei. DNA nicking occurred independently of caspase activation. The SET complex contains a 25-kDa Mg(2+)-dependent nuclease that degrades calf thymus DNA and plasmid DNA. Thus, GzmA activates a DNase (GzmA-activated DNase) within the SET complex to produce a novel form of DNA damage during cytotoxic T lymphocyte-mediated death.     

Viruses activate a genetically conserved cell death pathway in a unicellular organism

Given the importance of apoptosis in the pathogenesis of virus infections in mammals, we investigated the possibility that unicellular organisms also respond to viral pathogens by activating programmed cell death. The M1 and M2 killer viruses of Saccharomyces cerevisiae encode pore-forming toxins that were assumed to kill uninfected yeast cells by a nonprogrammed assault. However, we found that yeast persistently infected with these killer viruses induce a programmed suicide pathway in uninfected (nonself) yeast. The M1 virus-encoded K1 toxin is primarily but not solely responsible for triggering the death pathway. Cell death is mediated by the mitochondrial fission factor Dnm1/Drp1, the K+ channel Tok1, and the yeast metacaspase Yca1/Mca1 encoded by the target cell and conserved in mammals. In contrast, cell death is inhibited by yeast Fis1, a pore-forming outer mitochondrial membrane protein. This virus-host relationship in yeast resembles that of pathogenic human viruses that persist in their infected host cells but trigger programmed death of uninfected cells.