A caspase-related protease regulates apoptosis in yeast

Yeast can undergo cell death accompanied by cellular markers of apoptosis. However, orthologs of classical mammalian apoptosis regulators appeared to be missing from the yeast genome, challenging a common mechanism of yeast and mammalian apoptosis. Here we investigate Yor197w, a yeast protein with structural homology to mammalian caspases, and demonstrate caspase-like processing of the protein. Hydrogen peroxide treatment induces apoptosis together with a caspase-like enzymatic activity in yeast. This response is completely abrogated after disruption and strongly stimulated after overexpression of Yor197w. Yor197w also mediates the death process within chronologically aged cultures, pointing to a physiological role in elimination of overaged cells. We conclude that Yor197w indeed functions as a bona fide caspase in yeast and propose the name Yeast Caspase-1 (YCA1, gene YCA1).     

Investigation on cell death in the megagametophyte of Araucaria bidwillii Hook. post-germinated seeds

The megagametophyte of the Araucaria bidwillii seed is a storage tissue that surrounds and feeds the embryo. When all its reserves are mobilized, the megagametophyte degenerates as a no longer needed tissue. In this work we present a biochemical and a cytological characterization of the megagametophyte cell death. The TUNEL assay showed progressive DNA fragmentation throughout the post-germinative stages, while DNA electrophoretic analysis highlighted a smear as the predominant pattern of DNA degradation and internucleosomal DNA cleavage only for a minority of cells at late post-germinative stages. Cytological investigations at these stages detected profound changes in the size and morphology of the megagametophyte nuclei. By using in vitro assays, we were able to show a substantial increase in proteolytic activities, including caspase-like protease activities during the megagametophyte degeneration. Among the caspase-like enzymes, caspase 6- and 1-like proteases appeared to be the most active in the megagametophyte with a preference for acidic pH. On the basis of our results, we propose that the major pathway of cell death in the Araucaria bidwillii megagametophyte is necrosis; however, we do not exclude that some cells undergo developmental programmed cell death.     

Caspase-Like Activities Accompany Programmed Cell Death Events in Developing Barley Grains

Programmed cell death is essential part of development and cell homeostasis of any multicellular organism. We have analyzed programmed cell death in developing barley caryopsis at histological, biochemical and molecular level. Caspase-1, -3, -4, -6 and -8-like activities increased with aging of pericarp coinciding with abundance of TUNEL positive nuclei and expression of HvVPE4 and HvPhS2 genes in the tissue. TUNEL-positive nuclei were also detected in nucellus and nucellar projection as well as in embryo surrounding region during early caryopsis development. Quantitative RT-PCR analysis of micro-dissected grain tissues revealed the expression of HvVPE2a, HvVPE2b, HvVPE2d, HvPhS2 and HvPhS3 genes exclusively in the nucellus/nucellar projection. The first increase in cascade of caspase-1, -3, -4, -6 and -8-like activities in the endosperm fraction may be related to programmed cell death in the nucellus and nucellar projection. The second increase of all above caspase-like activities including of caspase-9-like was detected in the maturating endosperm and coincided with expression of HvVPE1 and HvPhS1 genes as well as with degeneration of nuclei in starchy endosperm and transfer cells. The distribution of the TUNEL-positive nuclei, tissues-specific expression of genes encoding proteases with potential caspase activities and cascades of caspase-like activities suggest that each seed tissue follows individual pattern of development and disintegration, which however harmonizes with growth of the other tissues in order to achieve proper caryopsis development.     

QUANTIFICATION OF THE RELATIVE ABUNDANCE OF THE TOXIC DINOFLAGELLATE,KARENIA BREVIS (DINOPHYTA), USING UNIQUE PHOTOPIGMENTS1

Diagnostic photopigment analysis is a useful tool for determining the presence and relative abundance of algal groups in natural phytoplankton assemblages. This approach is especially useful when a genus has a unique photopigment composition. The toxic dinoflagellate Karenia brevis (Davis) G. Hansen & Moestrup comb. nov. shares the diagnostic pigment gyroxanthin‐diester with only a few other dinoflagellates and lacks peridinin, one of the major diagnostic pigments of most dinoflagellate species. In this study, measurements of gyroxanthin‐diester and other diagnostic pigments of K. brevis were incorporated into the initial pigment ratio matrix of the chemical taxonomy program (CHEMTAX) to resolve the relative contribution of K. brevis biomass in mixed estuarine phytoplankton assemblages from Florida and Galveston Bay, Texas. The phytoplankton community composition of the bloom in Galveston Bay was calculated based on cell enumerations and biovolumetric measurements in addition to chl a‐specific photopigment estimates of biomass (HPLC and CHEMTAX). The CHEMTAX and biovolume estimates of the phytoplankton community structure were not significantly different and suggest that the HPLC–CHEMTAX approach provides reasonable estimates of K. brevis biomass in natural assemblages. The gyroxanthin‐diester content per cell of K. brevis from Galveston Bay was significantly higher than in K. brevis collected from the west coast of Florida. This pigment‐based approach provides a useful tool for resolving spatiotemporal distributions of phytoplankton in the presence of K. brevis blooms, when an appropriate initial ratio matrix is applied.     

A caspase-like decoy molecule enhances the activity of a paralogous caspase in the yellow fever mosquito,Aedes aegypti

Caspases are cysteine proteases that play critical roles in apoptosis and other key cellular processes. A mechanism of caspase regulation that has been described in mammals and nematodes involves caspase-like decoy molecules, enzymatically inactive caspase homologs that have arisen by gene duplication and acquired the ability to regulate other caspases. Caspase-like decoy molecules are not found in Drosophila melanogaster, raising the question of whether this type of caspase regulation exists in insects. Phylogenomic analysis of caspase genes from twelve Drosophila and three mosquito species revealed several examples of duplicated caspase homologs lacking critical catalytic residues, making them candidate caspase-like decoy molecules. One of these, CASPS18 from the mosquito Aedes aegypti, is a homolog of the D. melanogaster caspase Decay and contains substitutions in two critical amino acid positions, including the catalytic cysteine residue. As expected, CASPS18 lacked caspase activity, but co-expression of CASPS18 with a paralogous caspase, CASPS19, in mosquito cells or co-incubation of CASPS18 and CASPS19 recombinant proteins resulted in greatly enhanced CASPS19 activity. The discovery of potential caspase-like decoy molecules in several insect species opens new avenues for investigating caspase regulation in insects, particularly in disease vectors such as mosquitoes.     

Caspase-like activity in the seedlings of Pisum sativum eliminates weaker shoots during early vegetative development by induction of cell death

Activation of aspartate-specific cysteine proteases (caspases) plays a crucial role in programmed cell death (PCD) in animals. Although to date caspases have not been identified in plants, caspase-like activity was described in tobacco during a hypersensitive response to pathogens and in Arabidopsis and tomato cell cultures during chemical-induced PCD. Caspase-like activity was also detected in the course of plant development during petal senescence and endosperm PCD. It is shown here that caspase-like proteases play a crucial role in the developmental cell death of secondary shoots of pea seedlings that emerge after removal of the epicotyl. Caspase-like activity was induced in senescing secondary shoots, but not in dominant growing shoots, in contrast to the papain-like cysteine protease activity that was stronger in the dominant shoot. Revitalization of the senescing shoot by cutting of the dominant shoot reduced the caspase-like activity. Injection of caspase or cysteine protease inhibitors into the remaining epicotyl tissue suppressed the death of the secondary shoots, producing seedlings with two equal shoots. These results suggest that shoot selection in pea seedlings is controlled by PCD, through the activation of caspase-like proteases.     

Neurological illnesses associated with Florida red tide (Karenia brevis) blooms

Human respiratory and gastrointestinal illnesses can result from exposures to brevetoxins originating from coastal Florida red tide blooms, comprising the marine alga Karenia brevis (K. brevis). Only limited research on the extent of human health risks and illness costs due to K. brevis blooms has been undertaken to date. Because brevetoxins are known neurotoxins that are able to cross the blood-brain barrier, it is possible that exposure to brevetoxins may be associated with neurological illnesses. This study explored whether K. brevis blooms may be associated with increases in the numbers of emergency department visits for neurological illness. An exposure-response framework was applied to test the effects of K. brevis blooms on human health, using secondary data from diverse sources. After controlling for resident population, seasonal and annual effects, significant increases in emergency department visits were found specifically for headache (ICD-9 784.0) as a primary diagnosis during proximate coastal K. brevis blooms. In particular, an increased risk for older residents (≥55 years) was identified in the coastal communities of six southwest Florida counties during K. brevis bloom events. The incidence of headache associated with K. brevis blooms showed a small but increasing association with K. brevis cell densities. Rough estimates of the costs of this illness were developed for hypothetical bloom occurrences.     

Capacity to control oxidative stress-induced caspase-like activity determines the level of tolerance to salt stress in two contrasting maize genotypes

The response of two maize (Zea mays L.) genotypes, named GR (salt-tolerant) and SK (salt-sensitive), to salt stress (150 mM NaCl) was investigated under controlled environmental growth conditions. Genotype SK experienced more oxidative damage than the GR genotype when subjected to salt stress, which corresponded to higher O₂ ^? production rate and H₂O₂ content in the SK genotype than the GR genotype. Induction of caspase-like activity in response to salt stress was stronger in the SK genotype than in the GR genotype. On the other hand, induction of antioxidant enzyme activity to scavenge O₂ ^? and H₂O₂ in response to salt stress was weaker in the SK genotype than in the GR genotype. Consequently, the higher level of oxidative damage in the SK genotype in response to salt stress was manifested as more extensive cell death and biomass reduction in the SK genotype than it was in the GR genotype. Our results suggest that a direct relationship exists between salt stress-induced oxidative damage and cell death-inducing caspase-like activity, with tolerance to the salt stress being controlled by the efficiency of the plant antioxidant enzymes in limiting salt stress-induced oxidative damage and thus limiting cell death-inducing caspase-like activity.     

Autophagy and Cell-Death Proteases in Plants: Two Wheels of a Funeral Cart

Apoptosis is an evolutionarily young cell-death strategy evolved to disassemble animal cells through the action of the caspase family of proteases and phagocytic clearance. This strategy does not work in plants, which instead feature a phylogenetically older autophagic programmed cell death (PCD), as a bona fide type of cellular suicide. Recent work has begun to address the mechanistic roles for autophagic and proteolytic components, as well as their possible cooperation in plant PCD. A recent study has shown autophagosomal localization of a key cell-death proteolytic activity at the early stage of plant PCD. Here we focus on the relationship between autophagic and proteoloytic components in plant PCD at the cellular and organismal levels.

Addendum to:

Developmental Regulation of a VEIDase Caspase-Like Proteolytic Activity in Barley Caryopsis

M. Borén, A.S. Höglund, P. Bozhkov and C. Jansson

J Exp Bot; In press     

Poly(ADP-Ribose) Polymerase Is a Substrate Recognized by Two Metacaspases of Podospora anserina

The two metacaspases MCA1 and MCA2 of the fungal aging model organism Podospora anserina (PaMCA1 and PaMCA2, respectively) have previously been demonstrated to be involved in the control of programmed cell death (PCD) and life span. In order to identify specific pathways and components which are controlled by the activity of these enzymes, we set out to characterize them further. Heterologous overexpression in Escherichia coli of the two metacaspase genes resulted in the production of proteins which aggregate and form inclusion bodies from which the active protein has been recovered via refolding in appropriate buffers. The renaturated proteins are characterized by an arginine-specific activity and are active in caspase-like self-maturation leading to the generation of characteristic small protein fragments. Both activities are dependent on the presence of calcium. Incubation of the two metacaspases with recombinant poly(ADP-ribose) polymerase (PARP), a known substrate of mammalian caspases, led to the identification of PARP as a substrate of the two P. anserina proteases. Using double mutants in which P. anserina Parp (PaParp) is overexpressed and PaMca1 is either overexpressed or deleted, we provide evidence for in vivo degradation of PaPARP by PaMCA1. These results support the idea that the substrate profiles of caspases and metacaspases are at least partially overlapping. Moreover, they link PCD and DNA maintenance in the complex network of molecular pathways involved in aging and life span control.     

Essential requirement of cytochrome c release for caspase activation by procaspase-activating compound defined by cellular models

Mitochondrial cytochrome c (cyt. c) release and caspase activation are often impaired in tumors with Bcl-2 overexpression or Bax and Bak-defective status. Direct triggering of cell death downstream of Bax and Bak is an attractive strategy to kill such cancers. Small molecule compounds capable of direct caspase activation appear to be the best mode for killing such tumors. However, there is no precise model to screen such compounds. The currently employed cell-free systems possess the inherent drawback of lacking cellular contents and organelles that operate in integrating cell death signaling. We have developed highly refined cell-based approaches to validate direct caspase activation in cancer cells. Using this approach, we show that PAC-1 (first procaspase-activating compound), the first direct activator of procaspases identified in a cell-free system, in fact requires mitochondrial cyt. c release for triggering caspase activation similar to other antitumor agents. It can induce significant caspase activation and cell death in the absence of Bax and Bak, and in cells overexpressing Bcl-2 and Bcl-xL. This study for the first time defines precise criteria for the validation of direct caspase-activating compounds using specialized cellular models that is expected to accelerate the discovery of potential direct caspase activators.     

Evidence for programmed cell death and activation of specific caspase-like enzymes in the tomato fruit heat stress response

The tomato (Lycopersicon esculentum) fruit is the best available model to study the stress response of fleshy fruit. Programmed cell death (PCD) plays an important role in stress responses in mammals and plants. In this study, we provide evidence that PCD is triggered in the tomato fruit heat stress response by detection of the sequential diagnostic PCD events, including release of cytochrome c, activation of caspase-like proteases and the presence of TUNEL-positive nuclei. Investigating the time course of these events for 12 h after heat treatment indicated that cytochrome c release and caspase-like protease activation occurred rapidly and were consistent with the onset of DNA fragmentation. In addition, LEHDase and DEVDase enzymes were specifically activated in tomato fruit pericarp during the heat treatment and recovery time. There was no significant activation of YVADase or IETDase proteases. Preincubation of pericarp discs with the broad-spectrum, cell-permeable caspase inhibitor Z-VAD-FMK, suppressed heat-induced cell death measured by trypan blue, accompanied by a decrease in LEHDase and DEVDase activities. Gui-Qin Qu and Xiang Liu contributed equally to this work.     

植物细胞程序性死亡中的类胱天蛋白酶研究进展

胱天蛋白酶(caspases)在动物细胞程序性死亡(programmed cell death,PCD)的起始、执行以及信号转导阶段起着关键作用,目前在植物中也发现有类胱天蛋白酶(caspase-like proteases,CLPs)的存在,并确认液泡加工酶(VPEs)、metacaspases和丝氨酸内肽酶(sapases)具有CLPs的作用,并证实CLPs参与植物的生长发育、抗病性及胁迫诱导的细胞程序性死亡等.本文对植物CLPs活性、生化结构及生理作用等方面的研究进展进行综述,并与动物caspases进行比较,为今后CLPs活性调节、作用方式及其在植物细胞程序性死亡中的作用等方面的研究提供参考.     

Involvement of phospholipase D-related signal transduction in chemical-induced programmed cell death in tomato cell cultures

Phospholipase D (PLD) and its product phosphatidic acid (PA) are incorporated in a complex metabolic network in which the individual PLD isoforms are suggested to regulate specific developmental and stress responses, including plant programmed cell death (PCD). Despite the accumulating knowledge, the mechanisms through which PLD/PA operate during PCD are still poorly understood. In this work, the role of PLDα1 in PCD and the associated caspase-like proteolysis, ethylene and hydrogen peroxide (H₂O₂) synthesis in tomato suspension cells was studied. Wild-type (WT) and PLDα1-silenced cell lines were exposed to the cell death-inducing chemicals camptothecin (CPT), fumonisin B1 (FB1) and CdSO₄. A range of caspase inhibitors effectively suppressed CPT-induced PCD in WT cells, but failed to alleviate cell death in PLDα1-deficient cells. Compared to WT, in CPT-treated PLDα1 mutant cells, reduced cell death and decreased production of H₂O₂ were observed. Application of ethylene significantly enhanced CPT-induced cell death both in WT and PLDα1 mutants. Treatments with the PA derivative lyso-phosphatidic acid and mastoparan (agonist of PLD/PLC signalling downstream of G proteins) caused severe cell death. Inhibitors, specific to PLD and PLC, remarkably decreased the chemical-induced cell death. Taken together with our previous findings, the results suggest that PLDα1 contributes to caspase-like-dependent cell death possibly communicated through PA, reactive oxygen species and ethylene. The dead cells expressed morphological features of PCD such as protoplast shrinkage and nucleus compaction. The presented findings reveal novel elements of PLD/PA-mediated cell death response and suggest that PLDα1 is an important factor in chemical-induced PCD signal transduction.     

Caspase Inhibition in Select Olfactory Neurons Restores Innate Attraction Behavior in Aged Drosophila

Sensory and cognitive performance decline with age. Neural dysfunction caused by nerve death in senile dementia and neurodegenerative disease has been intensively studied; however, functional changes in neural circuits during the normal aging process are not well understood. Caspases are key regulators of cell death, a hallmark of age-related neurodegeneration. Using a genetic probe for caspase-3-like activity (DEVDase activity), we have mapped age-dependent neuronal changes in the adult brain throughout the lifespan of Drosophila. Spatio-temporally restricted caspase activation was observed in the antennal lobe and ellipsoid body, brain structures required for olfaction and visual place memory, respectively. We also found that caspase was activated in an age-dependent manner in specific subsets of Drosophila olfactory receptor neurons (ORNs), Or42b and Or92a neurons. These neurons are essential for mediating innate attraction to food-related odors. Furthermore, age-induced impairments of neural transmission and attraction behavior could be reversed by specific inhibition of caspase in these ORNs, indicating that caspase activation in Or42b and Or92a neurons is responsible for altering animal behavior during normal aging.     

Sorting out the role of reactive oxygen species during plant programmed cell death induced by ultraviolet-C overexposure

Previous studies have reported that light is required for activating Arabidopsis programmed cell death (PCD) induced by ultraviolet-C (UV-C) overexposure, and a caspase-like protease cleaving the caspase-3 substrate Asp-Glu-Val-Asp (DEVDase activity) is induced during this process. Our recent report has suggested that a quick burst of reactive oxygen species (ROS), which is mainly derived from mitochondria and chloroplasts, is induced in a light dependent manner during the early stages of UV-induced plant PCD. Concomitantly, the mitochondria undergo serious dysfunction including the MTP loss and the changes in distribution and mobility, which ultimately lead to apoptotic-cell death. Though some of signaling molecules have been elucidated in this type of plant cell death, the molecular mechanism about UV-induce Arabidopsis PCD is still poorly understood when comparing with the study of signaling pathways involved in animal cell apoptosis induced by UV. By using the Arabidopsis mesophyll protoplasts as a reference model, we have begun to shed light on the complexity of signaling pathway in UV-induced plant PCD. Recently we have tried to real-time detect the presence of caspase-like proteolytic activation, and to sort out the key role of ROS as well as to further assess the relationship between the ROS production and caspase-like activation in this type of plant apoptotic cell death.Key words: caspase-like activation, FRET, programmed cell death, reactive oxygen species, ultraviolet-CUltraviolet-C has been shown to be a very convenient trigger to induce PCD in plants and protoplasts.^1,2 Others have shown that UV induction of plant PCD requires light and that caspase-like proteolytic activation is induced in this process.¹ Our recent works have shown that ROS mainly localizing in mitochondria and chloroplasts are produced in a light dependent manner during the early stages of UV stress, and that ROS production and mitochondrial dysfunction play important roles during UV-induced Arabidopsis PCD (Fig. 1).² We also found that if the Arabidopsis plants, which were kept at light for 1 h after UV irradiation then were moved to the dark and kept for 60 h, showed no evident plant death phenomena (unpublished data), though burst of ROS has appeared after UV exposure and subsequent 1 h light irradiation.² In contrast, seedlings developed an obvious bleaching when kept in light for 60 h after UV treatment. These findings prompt us to carry out further investigations to dig out the role of ROS in the execution of this type of cell death, and to ask whether the produced ROS in the early stages is involved in the activation of caspase-like protease.Open in a separate windowFigure 1Hypothetical model of the signal transduction pathways in the plant programmed cell death induced by UV-C overexposure. After UV and light treatment a quick burst of ROS appear in the region of mitochondria and chloroplasts, then the mitochondria undergo functional dysfunction, which ultimately leads to cell death. Caspase-like activation and nucleus damage are also involved in the control of this type cell death. Solid line means the issues have been detected. Dotted line and question marks indicate events that have not been detected in this process. For detailed explanation, see the text.It has been reported that ROS is required for the release of cytochrome c (cyt c) and subsequent activation of caspase-like proteases during heat-shock induced plant PCD, and the addition of caspase inhibitors (zVAD-fmk or AC-DEVD-CHO) can prevent the degradation of cyt c and protect the plant cells from cell death.³ Thus these findings suggest that ROS can trigger the release of cyt c, but do not cause cell death, which requires caspase-like activation.³ Conversely, caspase inhibitors have also shown to effectively block the oxidative burst and the plant cell death induced by camptothecin incubation.⁴ These studies suggest the complex relationship between ROS production and caspase activation during execution of plant PCD event. The ROS production and the mitochondrial dysfunction during UV-induced plant PCD have been illustrated in our research. We have suggested the occurrence of MTP disruption during UV stress; however, whether cyt c is released from mitochondria has not been assessed (Fig. 1). The important roles of cyt c release and subsequent caspase activation have been suggested in various types of programmed cell death including mammal and plant cells.^3,5,6 It will be a very challenging work to detect whether cyt c is released from mitochondria and is involved in the caspase-like proteolytic activation, and to further elucidate the relationship between ROS production and caspase-like activation in UV-induced plant PCD (Fig. 1).The involvement of caspase-like proteases in the control of cell death activation in plants has been shown in various forms of plant PCD.⁷ Using synthetic fluorogenic caspase-3 substrate, DEVD cleavage activity was detected during UV or heat shock-induced apoptosis of plant cells, and caspase inhibitors were able to suppress these types of cell death.^1,3 Caspase-like activities have also been detected in plant hypersensitive response (HR) triggered by tobacco mosaic virus (TMV), or plant PCD induced by chemicals like camptothecin.^8,9 All these experiments suggest the existence of functional caspase proteolytic activity in plant cells undergoing PCD. However, most of these results are from in vitro analysis using synthetic fluorogenic substrates or synthetic peptide inhibitor to caspases, this demand us to further dig out the plant caspase encoding gene and to real-time detect the caspase-like activity in vivo.Another of our ongoing work is aiming to detect the caspase-3-like proteolytic activation in living plant cells during UV-induced plant PCD, which is achieved by using the fluorescence resonance energy transfer (FRET) technique. FRET is the phenomenon whereby a fluorescent molecule—the donor—transfers energy by a nonradiative (through space) mechanism to a neighboring chromophore - the acceptor.¹⁰ FRET as a powerful technique to monitor compartmentation and subcellular targeting as well as to visualize protein-protein interactions and proteases activity in living cells has gained increasing importance for biotechnological applications during the last few years.¹¹ During the past few years FRET technique has been successfully used to monitor interactions and distances between molecules in living plant cells.^12–14 Presently, we have constructed a recombinant caspase substrate to monitor caspase-3-like protease activation in single living plant protoplast in real time. This recombinant is composed of enhanced cyan fluorescence protein (ECFP) as the FRET donor and enhanced yellow fluorescence protein (EYFP) as the acceptor, linked by peptides containing the caspase-3 cleavage sequence, DEVD (ECFP-DEVD-EYFP) as the papers demonstrated. 15 Arabidopsis mesophyll protoplasts have been successfully transiently transfected with our recombinant plasmid for expression of ECFP-DEVD-EYFP fusion proteins under control of the CaMV 35S promoter according to a modified procedure (as described previously, ref. ¹⁶). Preliminary experimental results have proved the feasibility of this method to real-time detect the caspase-like activation in living plant cells during UV-induced plant PCD.Using this FRET probe, we may real-time detect the caspase-like activation during UV-induced plant PCD, and elucidate the relationship between ROS production and caspase-like activation as well as verify our hypothesis that whether ROS is necessary for the activation of caspase-like proteases during this process. So the role of ROS in the execution of this type cell death can be further investigated. These subsequent researches will certainly increase our knowledge about the signal transduction pathways in UV-induced Arabidopsis PCD.