Involvement of proteases in apoptosis

Genetically programmed (apoptotic) cell death plays a key role in cell and tissue homeostasis and in pathogenesis of various diseases. However, the mechanisms involved in apoptotic cell death are poorly understood. At present, the role of proteases in key events of apoptosis is intensively studied and discussed and the involvement of various proteolytic enzymes in the induction and development of the cell death is well-recognized. Proteases of various classes participating in apoptosis have been identified as well as some substrates of these proteases whose cleavage is critical to cell viability; specific protease inhibitors which prevent the cell death have been synthesized. This review summarizes new data on proteolytic enzymes involved in apoptosis and considers the mechanisms of activation of proteases upon induction of apoptosis and the pathways of their involvement in the cell death. The participation of nuclear proteolytic enzymes in the destabilization of chromatin structure and regulation of DNA fragmentation by endonucleases in apoptotic cells is discussed.     

Participation of proteolytic enzymes in the interaction of plants with phytopathogenic microorganisms

Different forms of participation of proteolytic enzymes in pathogenesis and plant defense are reviewed. Together with extracellular proteinases, phytopathogenic microorganisms produce specific effectors with proteolytic activity and are able to act on proteins inside the plant cell. In turn, plants use both extracellular and intracellular proteinases for defense against phytopathogenic microorganisms. Among the latter, a special role belongs to vacuolar processing enzymes (legumains), which perform the function of caspases in the plant cell.     

Proteomic analysis of extracellular ATP-regulated proteins identifies ATP synthase beta-subunit as a novel plant cell death regulator

Extracellular ATP is an important signal molecule required to cue plant growth and developmental programs, interactions with other organisms, and responses to environmental stimuli. The molecular targets mediating the physiological effects of extracellular ATP in plants have not yet been identified. We developed a well characterized experimental system that depletes Arabidopsis cell suspension culture extracellular ATP via treatment with the cell death-inducing mycotoxin fumonisin B1. This provided a platform for protein profile comparison between extracellular ATP-depleted cells and fumonisin B1-treated cells replenished with exogenous ATP, thus enabling the identification of proteins regulated by extracellular ATP signaling. Using two-dimensional difference in-gel electrophoresis and matrix-assisted laser desorption-time of flight MS analysis of microsomal membrane and total soluble protein fractions, we identified 26 distinct proteins whose gene expression is controlled by the level of extracellular ATP. An additional 48 proteins that responded to fumonisin B1 were unaffected by extracellular ATP levels, confirming that this mycotoxin has physiological effects on Arabidopsis that are independent of its ability to trigger extracellular ATP depletion. Molecular chaperones, cellular redox control enzymes, glycolytic enzymes, and components of the cellular protein degradation machinery were among the extracellular ATP-responsive proteins. A major category of proteins highly regulated by extracellular ATP were components of ATP metabolism enzymes. We selected one of these, the mitochondrial ATP synthase β-subunit, for further analysis using reverse genetics. Plants in which the gene for this protein was knocked out by insertion of a transfer-DNA sequence became resistant to fumonisin B1-induced cell death. Therefore, in addition to its function in mitochondrial oxidative phosphorylation, our study defines a new role for ATP synthase β-subunit as a pro-cell death protein. More significantly, this protein is a novel target for extracellular ATP in its function as a key negative regulator of plant cell death.     

A genome-based approach to study the mechanisms by which cell-wall type is defined and constructed by the collaborative actions of cell-wall-related enzymes

The cellulose/xyloglucan framework underpins the cell wall of most flowering plants, and the processes of construction and restructuring of this framework are considered to be mediated by several different classes of enzymes such as cellulose synthetases, β-1,4 glucanases, xyloglucan endotransglucosylases/hydrolases (XTH) and expansins. The Arabidopsis sequencing project has revealed that these enzymes are encoded, without exception, by large multi-gene families. Comprehensive expression-analyses of the XTH gene family, as assisted by real-time RT-PCR procedure, have revealed that each member of the gene family exhibits an expression profile distinct from the other members. The results obtained thus far support the idea that each member of the XTH gene family is regulated specifically by different sets of plant hormones and is committed to a certain specific process in a specific tissue, at specific stages of development. Based on these considerations, we advance a hypothesis that the cell wall in a certain cell-type is constructed, maintained and restructured by a series of collaborative actions of a set of enzymes that are characteristic of the cell-wall type. This hypothesis assumes that a master gene, specific for each cell type, conducts a set of enzymes required for certain types of cell-wall structure and, thereby, defines the cell-wall type and, hence, cell type, during the process of plant development. Electronic Publication     

植物类Caspase及其调控细胞程序性死亡的研究进展

植物细胞程序性死亡（PCD）在植物生长发育和逆境适应中发挥重要作用。半胱氨酸蛋白酶（caspase）调控动物PcD的启动、执行及信号转导。通过人工合成底物、动物caspase抑制剂等方法已证实在植物中存在类caspase,可分为metacas．pases、VPEs（vacuolar processing enzymes）和saspases等。本文综述了植物类caspase的种类、结构、定位、功能及其调控PCD的研究进展,提出植物PCD中类caspase作用的调控途径,为深入研究植物PCD提供参考。     

植物发育性细胞程序死亡的发生机制

细胞程序死亡是多细胞生物体在内源发育信号或外源环境信号作用下在特定时间和空间发生的细胞死亡过程, 在植物的生长发育过程中起着重要作用。该文介绍了植物细胞程序死亡类型的几种划分方法、植物发育性细胞程序死亡研究常用的实验体系, 并着重概述有关植物发育性细胞程序死亡发生机制的研究进展。     

Vacuolar processing enzyme: an executor of plant cell death

Apoptotic cell death in animals is regulated by cysteine proteinases called caspases. Recently, vacuolar processing enzyme (VPE) was identified as a plant caspase. VPE deficiency prevents cell death during hypersensitive response and cell death of limited cell layers at the early stage of embryogenesis. Because plants do not have macrophages, dying cells must degrade their materials by themselves. VPE plays an essential role in the regulation of the lytic system of plants during the processes of defense and development. VPE is localized in the vacuoles, unlike animal caspases, which are localized in the cytosol. Thus, plants might have evolved a regulated cellular suicide strategy that, unlike animal apoptosis, is mediated by VPE and the vacuoles.     

Identification of proteases that regulate erythrocyte rupture by the malaria parasite Plasmodium falciparum

Newly replicated Plasmodium falciparum parasites escape from host erythrocytes through a tightly regulated process that is mediated by multiple classes of proteolytic enzymes. However, the identification of specific proteases has been challenging. We describe here a forward chemical genetic screen using a highly focused library of more than 1,200 covalent serine and cysteine protease inhibitors to identify compounds that block host cell rupture by P. falciparum. Using hits from the library screen, we identified the subtilisin-family serine protease PfSU B1 and the cysteine protease dipeptidyl peptidase 3 (DPAP3) as primary regulators of this process. Inhibition of both DPAP3 and PfSUB1 caused a block in proteolytic processing of the serine repeat antigen (SERA) protein SERA5 that correlated with the observed block in rupture. Furthermore, DPAP3 inhibition reduced the levels of mature PfSUB1. These results suggest that two mechanistically distinct proteases function to regulate processing of downstream substrates required for efficient release of parasites from host red blood cells.     

植物细胞程序性死亡中的类caspases蛋白酶

细胞程序性死亡对于植物的正常生长发育及病理过程具有十分重要的生物学意义。现有的实验证据表明,细胞程序性死亡在动物和植物中有许多相似之处,但也各有特点。在植物中,VPEs、metacaspases和saspases等酶类在细胞程序性死亡过程中发挥了关键性作用。该文详细比较了动、植物细胞程序性死亡的差异,并阐述TVPEs、metacaspases和saspases三种类caspases蛋白酶在植物程序性细胞死亡中所起的作用。     

植物细胞程序性死亡中的类caspases 蛋白酶

细胞程序性死亡对于植物的正常生长发育及病理过程具有十分重要的生物学意义。现有的实验证据表明, 细胞程序性死亡在动物和植物中有许多相似之处, 但也各有特点。在植物中, VPEs、metacaspases和saspases 等酶类在细胞程序 性死亡过程中发挥了关键性作用。该文详细比较了动、植物细胞程序性死亡的差异, 并阐述了VPEs 、metacas pases 和saspases三种类caspases蛋白酶在植物程序性细胞死亡中所起的作用。     

Phytaspases: Aspartate-specific proteases involved in plant cell death

Structures and properties of the recently found aspartate-specific cell death-related plant proteases called phytaspases are reviewed and compared to those of caspases, animal apoptotic proteases. Caspases (cysteine-dependent proteases) and phytaspases (serine-dependent proteases) dramatically differ in structure, although manifest a similar substrate specificity and a play a similar role in the programmed cell death. Distinctions in the structural organization of animal and plant death proteases were shown to define differences in the regulation strategies of functioning of these proteolytic enzymes in the two kingdoms.     

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.     

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.     

Proteolytic cleavage during chemotherapy-induced apoptosis

Treatment with anticancer drugs sets into motion a morphologically and biochemically distinct type of cell death called apoptosis. Recent genetic and biochemical studies have suggested that proteases play a prominent role in the active phase of apoptotic cell death. Ongoing studies are aimed at identifying the proteases involved, the substrates that are cleaved, and the means by which the proteolytic process is regulated in nonapoptotic and apoptotic cells. The possibility that these findings will suggest new approaches to treating cancer and other diseases is discussed.     

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.     

Plant proteolytic enzymes: possible roles during programmed cell death

Proteolytic enzymes are known to be associated with developmentally programmed cell death during organ senescence and tracheary element differentiation. Recent evidence also links proteinases with some types of pathogen- and stress-induced cell suicide. The precise roles of proteinases in these and other plant programmed cell death processes are not understood, however. To provide a framework for consideration of the importance of proteinases during plant cell suicide, characteristics of the best-known proteinases from plants including subtilisin-type and papain-type enzymes, phytepsins, metalloproteinases and the 26S proteasome are summarized. Examples of serine, cysteine, aspartic, metallo- and threonine proteinases linked to animal programmed cell death are cited and the potential for plant proteinases to act as mediators of signal transduction and as effectors of programmed cell death is discussed.     

Role of proteolytic enzymes in the interaction of phytopathogenic microorganisms with plants

Various forms of participation of proteolytic enzymes in pathogenesis and defense in plants are reviewed. Along with extracellular proteinases, phytopathogenic microorganisms produce specific effectors having proteolytic activity and capable of acting on proteins inside plant cells. In turn, for defense against pathogens, plants use both extracellular and intracellular proteinases.     

AtFACE-2, a functional prenylated protein protease from Arabidopsis thaliana related to mammalian Ras-converting enzymes

Eukaryotic proteins containing a CAAX (A is aliphatic amino acid) C-terminal tetrapeptide sequence generally undergo a lipid modification, the addition of a prenyl group. Proteins that are modified by prenylation, such as Ras GTPases, can be subsequently modified by a proteolytic event that removes a C-terminal tripeptide (AAX). Two distinct proteases have been identified that are involved in the CAAX proteolytic step, FACE-1/Ste24 and FACE-2/Rce1. These proteases have different enzymatic properties, substrate specificities, and biological functions. However, a proposal has been made that plants lack a FACE-2/Rce1-type protease. Here, we describe the isolation of a cDNA from Arabidopsis thaliana that encodes a 311-aa protein with characteristics that are similar to the FACE-2/Rce1 group of enzymes. Northern blot analysis demonstrates widespread expression of this gene in plant tissues. Heterologous expression of the A. thaliana cDNA in yeast restores CAAX proteolytic activity to yeast lacking native CAAX proteases. The recombinant protein produced in this system displays an in vivo substrate specificity profile distinct from AtSte24 and cleaves a farnesylated CAAX tetrapeptide in vitro. These results provide evidence for the existence of a previously unsuspected plant FACE-2/Rce1 ortholog and support the evolutionary conservation of dual CAAX proteolytic systems in eukaryotes.