Sheathing the swords of death: Post-translational modulation of plant metacaspases

Plant metacaspases (MCPs) are conserved cysteine proteases that have been postulated as regulators of programmed cell death (PCD). Although MCPs have been proven to have PCD relevant functions in multiple species ranging from fungi to plants, how these proteases are modulated in vivo remains unclear. Aside from demonstrating that these proteases are distinct from metazoan caspases due to their different target site specificities, how these proteases are used to tightly regulate cell death progression is a key question that remains to be resolved. Some recent studies on the biochemical characteristics of type-II MCP activities in Arabidopsis may begin to shed additional light on this aspect. The in vitro catalytic activities of recombinant AtMC4, AtMC5 and AtMC8 are found to be Ca²⁺-dependent while recombinant AtMC9 is active under mildly acidic conditions and not dependent on stimulation by Ca²⁺. Alterations of cellular pH and Ca²⁺ concentration commonly occur during various stresses and may help to orchestrate differential activation of latent MCPs under these conditions. Recent peptide mapping for recombinant AtMC4 (also called Metacaspase-2d) followed by site-specific mutagenesis studies have revealed multiple potential self-cleavage sites with the identification of a conserved lysine residue (Lys-225) as the key position for enzyme function both in vitro and in vivo. The multiple self-cleavage sites in MCPs may also facilitate desensitization of these proteases and can provide a means for fine-tuning their proteolytic activities in order to achieve more sensitive control of downstream processes.     

Programmed cell death: a missing link is found

Two families of proteins have advanced our understanding of the molecular basis of programmed cell death (PCD) in animal cells - the caspases and Bcl-2-related proteins. While caspases lie at the heart of the death programme, Bcl-2-related proteins act as key intracellular regulators. Although there has been considerable progress in elucidating the biochemical functions of caspases, how Bcl-2-related proteins regulate caspase activation and thereby PCD, has remained a mystery. One key to resolving this mystery seems to lie with a new third family of proteins related to the Caenorhabditis elegans cell-death protein CED-4, which connects Bcl-2-related proteins to caspases. An important step in defining this new family has been made by the identification of a human CED-4 homologue.     

Death without caspases, caspases without death

Apoptosis is a conserved cell-death process displaying characteristic morphological and molecular changes including activation of caspase proteases. Recent work challenges the accepted roles of these proteases. New investigations in mice and the nematode Caenorhabditis elegans suggest that there could be caspase-independent pathways leading to cell death. In addition, another type of cell death displaying autophagic features might depend on caspases. Recent studies also indicate that caspase activation does not always lead to cell death and, instead, might be important for cell differentiation. Here, we review recent evidence for both the expanded roles of caspases and the existence of caspase-independent cell-death processes. We suggest that cellular context plays an important role in defining the consequences of caspase activation.     

Noncanonical cell death pathways act during Drosophila oogenesis

Programmed cell death (PCD) is a highly conserved process that occurs during development and in response to adverse conditions. In Drosophila, most PCDs require the genes within the H99 deficiency, the adaptor molecule Ark, and caspases. Here we investigate 10 cell death genes for their potential roles in two distinct types of PCD that occur in oogenesis: developmental nurse cell PCD and starvation-induced PCD. Most of the genes investigated were found to have little effect on late stage developmental PCD in oogenesis, although ark mutants showed a partial inhibition. Mid-stage starvation-induced germline PCD was found to be independent of the upstream activators and ark although it requires caspases, suggesting an apoptosome-independent mechanism of caspase activation in mid-oogenesis. These results indicate that novel pathways must control PCD in the ovary.     

Plant caspase-like proteases in plant programmed cell death

Programmed cell death (PCD) is a genetically-controlled disassembly of the cell. In animal systems, the central core execution switch for apoptotic PCD is the activation of caspases (Cysteine-containing Aspartate-specific proteases). Accumulating evidence in recent years suggests the existence of caspase-like activity in plants and its functional involvement in various types of plant PCD, although no functional homologs of animal caspases were identified in plant genome. In this mini-review, we will cover the recent results on the existence of plant caspase-like proteases and introduce major technologies used in detecting the activation of caspase-like proteases during plant PCD.Key words: caspase-like proteases, fluorescence resonance energy transfer, programmed cell death     

A tomato metacaspase gene is upregulated during programmed cell death in<Emphasis Type="Italic"> Botrytis cinerea</Emphasis>-infected leaves

Programmed cell death (PCD) in plant cells is often accompanied by biochemical and morphological hallmarks similar to those of animal apoptosis. However, orthologs of animal caspases, cysteinyl aspartate-specific proteases that constitute the core component of animal apoptosis, have not yet been identified in plants. Recent studies have revealed the presence of a family of genes encoding proteins with distant homology to mammalian caspases, designated metacaspases, in the Arabidopsis thaliana genome. Here, we describe the isolation of LeMCA1, a type-II metacaspase cDNA clone from tomato (Lycopersicon esculentum Mill.). BLAST analysis demonstrated that the LeMCA1 gene is located in close vicinity of several genes that have been linked with PCD. Southern analysis indicated the existence of at least one more metacaspase in the tomato genome. LeMCA1 mRNA levels rapidly increased upon infection of tomato leaves with Botrytis cinerea, a fungal pathogen that induces cell death in several plant species. LeMCA1 was not upregulated during chemical-induced PCD in suspension-cultured tomato cells.     

A plant caspase-like protease activated during the hypersensitive response

To test the hypothesis that caspase-like proteases exist and are critically involved in the implementation of programmed cell death (PCD) in plants, a search was undertaken for plant caspases activated during the N gene-mediated hypersensitive response (HR; a form of pathogen-induced PCD in plants) in tobacco plants infected with Tobacco mosaic virus (TMV). For detection, characterization, and partial purification of a tobacco caspase, the Agrobacterium tumefaciens VirD2 protein, shown here to be cleaved specifically at two sites (TATD and GEQD) by human caspase-3, was used as a target. In tobacco leaves, specific proteolytic processing of the ectopically produced VirD2 derivatives at these sites was found to occur early in the course of the HR triggered by TMV. A proteolytic activity capable of specifically cleaving the model substrate at TATD was partially purified from these leaves. A tetrapeptide aldehyde designed and synthesized on the basis of the elucidated plant caspase cleavage site prevented fragmentation of the substrate protein by plant and human caspases in vitro and counteracted TMV-triggered HR in vivo. Therefore, our data provide a characterization of caspase-specific protein fragmentation in apoptotic plant cells, with implications for the importance of such activity in the implementation of plant PCD.     

The Drosophila caspases Strica and Dronc function redundantly in programmed cell death during oogenesis

Programmed cell death (PCD) in the Drosophila ovary occurs either during mid-oogenesis, resulting in degeneration of the entire egg chamber or during late oogenesis, to facilitate the development of the oocyte. PCD during oogenesis is regulated by mechanisms different from those that control cell death in other Drosophila tissues. We have analyzed the role of caspases in PCD of the female germline by examining caspase mutants and overexpressing caspase inhibitors. Imprecise P-element excision was used to generate mutants of the initiator caspase strica. While null mutants of strica or another initiator caspase, dronc, display no ovary phenotype, we find that strica exhibits redundancy with dronc, during both mid- and late oogenesis. Ovaries of double mutants contain defective mid-stage egg chambers similar to those reported previously in dcp-1 mutants, and mature egg chambers with persisting nurse cell nuclei. In addition, the effector caspases drice and dcp-1 also display redundant functions during late oogenesis, resulting in persisting nurse cell nuclei. These findings indicate that caspases are required for nurse cell death during mid-oogenesis, and participate in developmental nurse cell death during late oogenesis. This reveals a novel pathway of cell death in the ovary that utilizes strica, dronc, dcp-1 and drice, and importantly illustrates strong redundancy among the caspases.     

The lace plant: a novel model system to study plant proteases during developmental programmed cell death in vivo

Programmed cell death (PCD) plays a major role in plant development and defense throughout the plant kingdom. Within animal systems, it is well accepted that caspases play a major role in the PCD process, although no true caspases have yet to be identified in plants. Despite this, vast amounts of evidence suggest the existence of caspase-like proteases in plants. The lace plant (Aponogeton madagascariensis) forms perforations in a predictable pattern between longitudinal and transverse veins over its entire leaf surface via PCD. Due to the thin nature of the leaf, allowing for long-term live cell imaging, a perfected method for sterile culture, as well as the feasibility of pharmacological experiments, the lace plant provides an excellent model to study developmental PCD. In this review, we report the suitability of the lace plant as a novel organism to study proteases in vivo during developmentally regulated cell death.     

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.     

Metacaspase-8 modulates programmed cell death induced by ultraviolet light and H2O2 in Arabidopsis

Programmed cell death (PCD) is a genetically controlled cell death that is regulated during development and activated in response to environmental stresses or pathogen infection. The degree of conservation of PCD across kingdoms and phylum is not yet clear; however, whereas caspases are proteases that act as key components of animal apoptosis, plants have no orthologous caspase sequences in their genomes. The discovery of plant and fungi metacaspases as proteases most closely related to animal caspases led to the hypothesis that metacaspases are the functional homologues of animal caspases in these organisms. Arabidopsis thaliana has nine metacaspase genes, and so far it is unknown which members of the family if any are involved in the regulation of PCD. We show here that metacaspase-8 (AtMC8) is a member of the gene family strongly up-regulated by oxidative stresses caused by UVC, H(2)O(2), or methyl viologen. This up-regulation was dependent of RCD1, a mediator of the oxidative stress response. Recombinant metacaspase-8 cleaved after arginine, had a pH optimum of 8, and complemented the H(2)O(2) no-death phenotype of a yeast metacaspase knock-out. Overexpressing AtMC8 up-regulated PCD induced by UVC or H(2)O(2), and knocking out AtMC8 reduced cell death triggered by UVC and H(2)O(2) in protoplasts. Knock-out seeds and seedlings had an increased tolerance to the herbicide methyl viologen. We suggest that metacaspase-8 is part of an evolutionary conserved PCD pathway activated by oxidative stress.     

A plant alternative to animal caspases: subtilisin-like proteases

Activities displaying caspase cleavage specificity have been well documented in various plant programmed cell death (PCD) models. However, plant genome analyses have not revealed clear orthologues of caspase genes, indicating that enzyme(s) structurally unrelated yet possessing caspase specificity have functions in plant PCD. Here, we review recent data showing that some caspase-like activities are attributable to the plant subtilisin-like proteases, saspases and phytaspases. These proteases hydrolyze a range of tetrapeptide caspase substrates following the aspartate residue. Data obtained with saspases implicate them in the proteolytic degradation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) during biotic and abiotic PCD, whereas phytaspase overproducing and silenced transgenics provide evidence that phytaspase regulates PCD during both abiotic (oxidative and osmotic stresses) and biotic (virus infection) insults. Like caspases, phytaspases and saspases are synthesized as proenzymes, which are autocatalytically processed to generate a mature enzyme. However, unlike caspases, phytaspases and saspases appear to be constitutively processed and secreted from healthy plant cells into the intercellular space. Apoplastic localization presumably prevents enzyme-mediated protein fragmentation in the absence of PCD. In response to death-inducing stimuli, phytaspase has been shown to re-localize to the cell interior. Thus, plant PCD-related proteases display both common (D-specific protein fragmentation during PCD) and distinct (enzyme structure and activity regulation) features with animal PCD-related proteases.     

Beyond apoptosis: nonapoptotic cell death in physiology and disease.

Apoptosis is a morphologically defined form of programmed cell death (PCD) that is mediated by the activation of members of the caspase family. Analysis of death-receptor signaling in lymphocytes has revealed that caspase-dependent signaling pathways are also linked to cell death by nonapoptotic mechanisms, indicating that apoptosis is not the only form of PCD. Under physiological and pathological conditions, cells demonstrate a high degree of flexibility in cell-death responses, as is reflected in the existence of a variety of mechanisms, including necrosis-like PCD, autophagy (or type II PCD), and accidental necrosis. In this review, we discuss recent data suggesting that canonical apoptotic pathways, including death-receptor signaling, control caspase-dependent and -independent cell-death pathways.     

Caspase inhibitors affect the kinetics and dimensions of tracheary elements in xylogenic Zinnia (<Emphasis Type="Italic">Zinnia elegans</Emphasis>) cell cultures

Background  

The xylem vascular system is composed of fused dead, hollow cells called tracheary elements (TEs) that originate through trans-differentiation of root and shoot cambium cells. TEs undergo autolysis as they differentiate and mature. The final stage of the formation of TEs in plants is the death of the involved cells, a process showing some similarities to programmed cell death (PCD) in animal systems. Plant proteases with functional similarity to proteases involved in mammalian apoptotic cell death (caspases) are suggested as an integral part of the core mechanism of most PCD responses in plants, but participation of plant caspase-like proteases in TE PCD has not yet been documented.     

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.     

New types of metacaspases in phytoplankton reveal diverse origins of cell death proteases

Metacaspases are evolutionarily distant homologs of caspases that are found outside the metazoan and are known to have key roles in programmed cell death (PCD). Two types of metacaspases (types I and II) have been defined in plants based on their domain structures; these have similarities to metazoan ‘initiator'' and ‘executioner'' caspases. However, we know little about metacaspases in unicellular organisms and even less about their roles in cell death. We identified a novel group of metacaspases in sequenced phytoplanktonic protists that show domain architectures distinct from either type I or II enzymes; we designate them as type III. Type III metacaspases exhibit a rearrangement of domain structures between N- and C-terminus. In addition, we found a group of metacaspase-like proteases in phytoplankton that show sequence homology with other metacaspases, but defy classification in conventional schemes. These metacaspase-like proteases exist in bacteria alongside a variant of type I metacaspases and we propose these bacterial metacaspases are the origins of eukaryotic metacaspases. Type II and III metacaspases were not detected in bacteria and they might be variants of bacterial type I metacaspases that evolved in plants and phytoplanktonic protists, respectively, during the establishment of plastids through the primary and secondary endosymbiotic events. A complete absence of metacaspases in protists that lost plastids, such as oömycetes and ciliates indicates the gene loss during the plastid-to-nucleus gene transfer. Taken together, our findings suggest endosymbiotic gene transfer (EGT) is a key mechanism resulting in the evolutionary diversity of cell death proteases.     

Plant phytaspases and animal caspases: structurally unrelated death proteases with a common role and specificity

Proteases with an aspartate cleavage specificity are known to contribute to programmed cell death (PCD) in animals and plants. In animal cells this proteolytic activity belongs to caspases, a well-characterized family of cysteine-dependent death proteases. Plants, however, lack caspase homologs and thus the origin of this type of proteolytic activity in planta was poorly understood. Here, we review recent data demonstrating that a plant serine-dependent protease, phytaspase, shares cleavage specificity and a role in PCD analogous to that of caspases. However, unlike caspases, regulation of phytaspase-mediated cleavage of intracellular target proteins appears to be attained not at the level of proenzyme processing/activation, which occurs, in the case of phytaspase, autocatalytically and constitutively. Rather, the mature phytaspase is excluded from healthy cells into the apoplast and is allowed to re-enter cells upon the induction of PCD. Thus, PCD-related proteases in animals and plants display both common features and important distinctions.     

The serine protease inhibitor TLCK attenuates intrinsic death pathways in neurons upstream of mitochondrial demise

It is well-established that activation of proteases, such as caspases, calpains and cathepsins are essential components in signaling pathways of programmed cell death (PCD). Although these proteases have also been linked to mechanisms of neuronal cell death, they are dispensable in paradigms of intrinsic death pathways, e.g. induced by oxidative stress. However, emerging evidence implicated a particular role for serine proteases in mechanisms of PCD in neurons. Here, we investigated the role of trypsin-like serine proteases in a model of glutamate toxicity in HT-22 cells. In these cells glutamate induces oxytosis, a form of caspase-independent cell death that involves activation of the pro-apoptotic protein BH3 interacting-domain death agonist (Bid), leading to mitochondrial demise and ensuing cell death. In this model system, the trypsin-like serine protease inhibitor Nα-tosyl-l-lysine chloromethyl ketone hydrochloride (TLCK) inhibited mitochondrial damage and cell death. Mitochondrial morphology alterations, the impairment of the mitochondrial membrane potential and ATP depletion were prevented and, moreover, lipid peroxidation induced by glutamate was completely abolished. Strikingly, truncated Bid-induced cell death was not affected by TLCK, suggesting a detrimental activity of serine proteases upstream of Bid activation and mitochondrial demise. In summary, this study demonstrates the protective effect of serine protease inhibition by TLCK against oxytosis-induced mitochondrial damage and cell death. These findings indicate that TLCK-sensitive serine proteases play a crucial role in cell death mechanisms upstream of mitochondrial demise and thus, may serve as therapeutic targets in diseases, where oxidative stress and intrinsic pathways of PCD mediate neuronal cell death.     

Phytaspase,a relocalisable cell death promoting plant protease with caspase specificity

Caspases are cysteine‐dependent proteases and are important components of animal apoptosis. They introduce specific breaks after aspartate residues in a number of cellular proteins mediating programmed cell death (PCD). Plants encode only distant homologues of caspases, the metacaspases that are involved in PCD, but do not possess caspase‐specific proteolytic activity. Nevertheless, plants do display caspase‐like activities indicating that enzymes structurally distinct from classical caspases may operate as caspase‐like proteases. Here, we report the identification and characterisation of a novel PCD‐related subtilisin‐like protease from tobacco and rice named phytaspase (plant aspartate‐specific protease) that possesses caspase specificity distinct from that of other known caspase‐like proteases. We provide evidence that phytaspase is synthesised as a proenzyme, which is autocatalytically processed to generate the mature enzyme. Overexpression and silencing of the phytaspase gene showed that phytaspase is essential for PCD‐related responses to tobacco mosaic virus and abiotic stresses. Phytaspase is constitutively secreted into the apoplast before PCD, but unexpectedly is re‐imported into the cell during PCD providing insights into how phytaspase operates.