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.     

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.     

Substrate Specificity and Possible Heterologous Targets of Phytaspase,a Plant Cell Death Protease

Plants lack aspartate-specific cell death proteases homologous to animal caspases. Instead, a subtilisin-like serine-dependent plant protease named phytaspase shown to be involved in the accomplishment of programmed death of plant cells is able to hydrolyze a number of peptide-based caspase substrates. Here, we determined the substrate specificity of rice (Oryza sativa) phytaspase by using the positional scanning substrate combinatorial library approach. Phytaspase was shown to display an absolute specificity of hydrolysis after an aspartic acid residue. The preceding amino acid residues, however, significantly influence the efficiency of hydrolysis. Efficient phytaspase substrates demonstrated a remarkable preference for an aromatic amino acid residue in the P3 position. The deduced optimum phytaspase recognition motif has the sequence IWLD and is strikingly hydrophobic. The established pattern was confirmed through synthesis and kinetic analysis of cleavage of a set of optimized peptide substrates. An amino acid motif similar to the phytaspase cleavage site is shared by the human gastrointestinal peptide hormones gastrin and cholecystokinin. In agreement with the established enzyme specificity, phytaspase was shown to hydrolyze gastrin-1 and cholecystokinin at the predicted sites in vitro, thus destroying the active moieties of the hormones.     

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.     

Arabidopsis metacaspase 2d is a positive mediator of cell death induced during biotic and abiotic stresses

Cysteine proteases such as caspases play important roles in programmed cell death (PCD) of metazoans. Plant metacaspases (MCPs), a family of cysteine proteases structurally related to caspases, have been hypothesized to be ancestors of metazoan caspases, despite their different substrate specificity. Arabidopsis thaliana contains six type II MCP genes (AtMCP2a-f). Whether and how these individual members are involved in controlling PCD in plants remains largely unknown. Here we investigated the function and regulation of AtMCP2d, the predominant and constitutively expressed member of type II MCPs, in stress-inducible PCD. Two AtMCP2d mutants (mcp2d-1 and mcp2d-3) exhibited reduced sensitivity to PCD-inducing mycotoxin fumonisin B1 as well as oxidative stress inducers, whereas AtMCP2d over-expressors were more sensitive to these agents, and exhibited accelerated cell-death progression. We found that AtMCP2d exclusively localizes to the cytosol, and its accumulation and self-processing patterns were age-dependent in leaves. Importantly, active proteolytic processing of AtMCP2d proteins dependent on its catalytic activity was observed in mature leaves during mycotoxin-induced cell death. We also found that mcp2d-1 leaves exhibited reduced cell death in response to Pseudomonas syringae carrying avirulent gene avrRpt2, and that self-processing of AtMCP2d was also detected in wild-type leaves in response to this pathogen. Furthermore, increases in processed AtMCP2d proteins were found to correlate with conditional cell-death induction in two lesion-mimic mutants (cpr22 and ssi4) that exhibit spontaneous cell-death phenotypes. Taken together, our data strongly suggest that AtMCP2d plays a positive regulatory role in biotic and abiotic stress-induced PCD.     

Programmed cell death in plants

The modern concepts of programmed cell death (PCD) in plants are reviewed as compared to PCD (apoptosis) in animals. Special attention is focused on considering the potential mechanisms of implementation of this fundamental biological process and its participants. In particular, the proteolytic enzymes involved in PCD in animals (caspases) and plants (phytaspases) are compared. Emphasis is put on elucidation of both common features and substantial differences of PCD implementation in plants and animals.     

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.     

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.     

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     

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.     

Proteases and caspase-like activity in the yeast Saccharomyces cerevisiae

A variety of proteases have been implicated in yeast PCD (programmed cell death) including the metacaspase Mca1 and the separase Esp1, the HtrA-like serine protease Nma111, the cathepsin-like serine carboxypeptideases and a range of vacuolar proteases. Proteasomal activity is also shown to have an important role in determining cell fate, with both pro- and anti-apoptotic roles. Caspase 3-, 6- and 8-like activities are detected upon stimulation of yeast PCD, but not all of this activity is associated with Mca1, implicating other proteases with caspase-like activity in the yeast cell death response. Global proteolytic events that accompany PCD are discussed alongside a consideration of the conservation of the death-related degradome (both at the level of substrate choice and cleavage site). The importance of both gain-of-function changes in the degradome as well as loss-of-function changes are highlighted. Better understanding of both death-related proteases and their substrates may facilitate the design of future antifungal drugs or the manipulation of industrial yeasts for commercial exploitation.     

Catalytic properties of the caspases

Caspase stands for cysteine-dependent aspartate specific protease, and is a term coined to define proteases related to interleukin 1beta converting enzyme and CED-3.1 Thus their enzymatic properties are governed by a dominant specificity for substrates containing Asp, and by the use of a Cys side-chain for catalyzing peptide bond cleavage. The use of a Cys side chain as a nucleophile during peptide bond hydrolysis is common to several protease families. However, the primary specificity for Asp turns out to be very rare among protease families throughout biotic kingdoms. Of all known mammalian proteases only the caspase activator granzyme B, a serine protease, has the same primary specificity. In addition to this unusual primary specificity, caspases are remarkable in that certain of their zymogens have intrinsic proteolytic activity. This latter property is essential to trigger the proteolytic pathways that lead to apoptosis. Here we review the known enzymatic properties of the caspases and their zymogens within the broad context of structure:mechanism:activity relationships of proteases in general.     

植物Metacaspase研究进展

过敏性坏死反应是植物的一种重要的抗病机制, 类似于动物细胞凋亡, 它是一种程序性细胞死亡(programmed cell death, PCD)过程。目前, 已经确定半胱天冬蛋白酶(caspase)在动物PCD过程中起核心作用。在植物中, 尚未发现其直系同源蛋白, 但是有一类与其结构相似的蛋白酶, 称为metacaspase。在植物不同的PCD过程中, 有的依赖于metacaspase, 而有的则不依赖于该类蛋白酶。目前对metacaspase的结构和功能已有了初步的研究, 对其深入的研究则进展缓慢, 其具体的生物学功能和在PCD信号路径中的定位有待进一步探索。     

Programmed cell death

This paper reviews data on programmed cell death (apoptosis) in animals and plants. Necrosis is a pathological scenario of cell death, which entails an inflammatory response in animal tissues. Apoptosis results in the disintegration of animal/plant cells into membrane vesicles enclosing the intracellular content, which are thereupon engulfed by adjacent or specialized cells (phagocytes) in animals. Plants lack such specialized cells, and plant cell walls prevent phagocytosis. The paper considers the main molecular mechanisms of apoptosis in animals and the pathways of activation of caspases, evolutionarily conserved cysteine proteases. A self-contained section concerns itself with the process of programmed cell death (PCD) in microorganisms including: 1) cell death in the myxomycete Dictyostelium discoideum and the parasitic flagellate Trypanosoma cruzi; 2) PCD in genetically manipulated yeast expressing the proapoptotic Bax and Bak proteins; 3) the death of a part of a prokaryotic cell population upon the depletion of nutrient resources or under stress; 4) the elimination of cells after a loss of a plasmid encoding a stable cytotoxic agent in combination with an unstable antidote; and 5) PCD in phage-infected bacterial cells.     

Caspase-like proteases and their role in programmed cell death in plants

The investigations performed over recent few years have proved the existence of caspase-like proteases in plants. Three groups of caspase-like proteases: metacaspases, legumain family proteases (VPEs) and saspases have been identified and characterized in plants so far. A considerable amount of evidence supports the role of these enzymes in programmed cell death (PCD) occurring during plant development, their organ senescence as well as hypersensitive response (HR) after pathogen attack. Current knowledge of these enzyme molecular and biochemical structures is summarized in the paper. The homology of caspase-like proteases to animal caspases has been also indicated. Some future perspectives of research concerning the signal pathway during PCD, the regulation of activity and mode of action of these proteases are presented in the article.     

Metacaspases

Metacaspases are cysteine-dependent proteases found in protozoa, fungi and plants and are distantly related to metazoan caspases. Although metacaspases share structural properties with those of caspases, they lack Asp specificity and cleave their targets after Arg or Lys residues. Studies performed over the past 10 years have demonstrated that metacaspases are multifunctional proteases essential for normal physiology of non-metazoan organisms. This article provides a comprehensive overview of the metacaspase function and molecular regulation during programmed cell death, stress and cell proliferation, as well as an analysis of the first metacaspase-mediated proteolytic pathway. To prevent further misapplication of caspase-specific molecular probes for measuring and inhibiting metacaspase activity, we provide a list of probes suitable for metacaspases.     

A cellular suicide strategy of plants: vacuole-mediated cell death

Programmed cell death (PCD) occurs in animals and plants under various stresses and during development. Recently, vacuolar processing enzyme (VPE) was identified as an executioner of plant PCD. VPE is a cysteine protease that cleaves a peptide bond at the C-terminal side of asparagine and aspartic acid. VPE exhibited enzymatic properties similar to that of a caspase, which is a cysteine protease that mediates the PCD pathway in animals, although there is limited sequence identity between the two enzymes. VPE and caspase-1 share several structural properties: the catalytic dyads and three amino acids forming the substrate pockets (Asp pocket) are conserved between VPE and caspase-1. In contrast to such similarities, subcellular localizations of these proteases are completely different from each other. VPE is localized in the vacuoles, while caspases are localized in the cytosol. VPE functions as a key molecule of plant PCD through disrupting the vacuole in pathogenesis and development. Cell death triggered by vacuolar collapse is unique to plants and has not been seen in animals. Plants might have evolved a VPE-mediated vacuolar system as a cellular suicide strategy.     

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.     

Death substrates come alive

Interleukin 1β-converting enzyme (ICE)-like proteases (caspases) play an important role in programmed cell death (apoptosis), and elucidating the consequences of their proteolytic activity is central to our understanding of the molecular mechanisms of cell death. Diverse structural and regulatory proteins and enzymes, including protein kinase Cδ, the retinoblastoma protein (a protein involved in cell survival), the DNA repair enzyme DNA-dependent protein kinase and the nuclear lamins, undergo specific and limited endoproteolytic cleavage by various caspases during apoptosis. Since individual caspases can cleave multiple substrates, the consequences of cleavage of only a single substrate are still poorly understood. Nevertheless, proteolytic activation of protein kinase Cδ may be an important early step in the cell death pathway, and cleavage of the retinoblastoma protein could suppress its cell survival function, whereas proteolytic inactivation of DNA repair enzymes might compromise the ability of the cell to reverse DNA fragmentation. On the other hand, cleavages of nuclear and cytoplasmic structural proteins (e.g. the lamins and Gas2) appear to be required for or contribute to the dramatic rearrangements in cellular architecture that are necessary for the completion of the cell death process. An emerging theme is that parallel and sequential proteolytic activation and inactivation of key protein substrates occurs during the multiple steps of apoptosis.