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.     

Prostaglandin-induced programmed cell death in Trypanosoma brucei involves oxidative stress

Recently, we reported the induction of a programmed cell death (PCD) in bloodstream forms of Trypanosoma brucei by prostaglandin D(2) (PGD(2)). As this prostanoid is readily metabolized in the presence of albumin, we were prompted to investigate if PGD(2) metabolites rather than PGD(2) itself are responsible for the observed PCD. In fact, J series metabolites, especially PGJ(2) and Delta(12)PGJ(2), were able to induce PCD more efficiently than PGD(2). However, the stable PGD(2) analog 17phenyl-trinor-PGD(2) led to the same phenotype as the natural PGD(2), indicating that the latter induces PCD as well. Interestingly, the intracellular reactive oxygen species (ROS) level increased significantly under J series metabolites treatment and, incubation with N-acetyl-L-cysteine or glutathione reduced ROS production and cell death significantly. We conclude that PGJ(2) and Delta(12)PGJ(2) formation within the serum represents a mechanism to amplify PGD(2)-induced PCD in trypanosomes via ROS production.     

Mitochondrial programmed cell death pathways in yeast

Whether or not yeast cell death is altruistic, apoptotic, or otherwise analogous to programmed cell death in mammals is controversial. However, growing attention to cell death mechanisms in yeast has produced several new papers that make a case for ancient origins of programmed death involving mitochondrial pathways conserved between yeast and mammals.     

The metabolism beyond programmed cell death in yeast

A cell's reaction to any change in the endogenous or exogenous conditions often involves a complex response that eventually either leads to cell adaptation and survival or to the initiation and execution of (programmed) cell death. The molecular decision whether to live or die, while depending on a cell's genome, is fundamentally influenced by its actual metabolic status. Thus, the collection of all metabolites present in a biological system at a certain time point (the so-called metabolome) defines its physiological, developmental and pathological state and determines its fate during changing and stressful conditions. The budding yeast Saccharomyces cerevisiae is a unicellular organism that allows to easily modify and monitor conditions affecting the cell's metabolome, for instance through a simple change of the nutrition source. Such changes can be used to mimic and study (patho)physiological scenarios, including caloric restriction and longevity, the Warburg effect in cancer cells or changes in mitochondrial mass affecting cell death. In addition, disruption of single genes or generation of respiratory deficiency (via abrogation of mitochondrial DNA) assists in revealing connections between metabolism and apoptosis. In this minireview, we discuss recent studies using the potential of the yeast model to provide new insights into the processes of stress defense, cell death and longevity.     

Mitochondria,oxidative stress and cell death

In addition to the well-established role of the mitochondria in energy metabolism, regulation of cell death has recently emerged as a second major function of these organelles. This, in turn, seems to be intimately linked to their role as the major intracellular source of reactive oxygen species (ROS), which are mainly generated at Complex I and III of the respiratory chain. Excessive ROS production can lead to oxidation of macromolecules and has been implicated in mtDNA mutations, ageing, and cell death. Mitochondria-generated ROS play an important role in the release of cytochrome c and other pro-apoptotic proteins, which can trigger caspase activation and apoptosis. Cytochrome c release occurs by a two-step process that is initiated by the dissociation of the hemoprotein from its binding to cardiolipin, which anchors it to the inner mitochondrial membrane. Oxidation of cardiolipin reduces cytochrome c binding and results in an increased level of “free” cytochrome c in the intermembrane space. Conversely, mitochondrial antioxidant enzymes protect from apoptosis. Hence, there is accumulating evidence supporting a direct link between mitochondria, oxidative stress and cell death.     

Expanding roles of programmed cell death in mammalian neurodevelopment

Programmed cell death is an orchestrated form of cell death in which cells are actively involved in their own demise. During neural development in mammals, many progenitor cells, immature cells or differentiated cells undergo the most clearly characterized type of cell death, apoptosis. Several pathways of apoptosis have been linked to neural development, but according to the numerous and striking phenotypes observed when apoptotic genes are inactivated, the mitochondrial death-route is the most important pathway in this context. Here, we discuss the relative importance of pro-growth/pro-death factors in the control of neural tissue development. We also discuss the impact of studying programmed cell death in development in order to better understand the basis of several human diseases and embryonic defects of the nervous system.     

Life and death decisions: the role of the IAPs in modulating programmed cell death

Multicellular organisms have evolved elaborate signal transduction pathways for maintaining homeostasis through the control of cell proliferation and death. The recent surge of interest in the regulation of programmed cell death has led to the rapid identification of many proteins involved in controlling and executing apoptosis. The inhibitors of apoptosis proteins (IAPs) constitute a family of highly conserved death suppressing proteins that were first identified in baculoviruses, and that has recently expanded to include at least two homologues in Drosophila melanogaster and four in rodents and humans. In this article we review the current state of IAP research. Two of the IAPs, HIAP-1 and HIAP-2, have been placed within the TNFα induced cell death pathway which involves two receptors for TNFα and multiple, overlapping signal transduction proteins. A third, X-linked gene termed XIAP, is ubiquitously expressed and appears to have a broad range of suppressor activity to a variety of apoptotic triggers. The fourth member, NAIP, has been identified as the protein product of a candidate gene for the inherited neuromuscular disorder, spinal muscular atrophy (SMA). The neuroprotective activity of NAIP in an in vivo model of cerebral ischemia has also been demonstrated. This revised version was published online in November 2006 with corrections to the Cover Date.     

Old yellow enzymes protect against acrolein toxicity in the yeast Saccharomyces cerevisiae

Acrolein is a ubiquitous reactive aldehyde which is formed as a product of lipid peroxidation in biological systems. In this present study, we screened the complete set of viable deletion strains in Saccharomyces cerevisiae for sensitivity to acrolein to identify cell functions involved in resistance to reactive aldehydes. We identified 128 mutants whose gene products are localized throughout the cell. Acrolein-sensitive mutants were distributed among most major biological processes but particularly affected gene expression, metabolism, and cellular signaling. Surprisingly, the screen did not identify any antioxidants or similar stress-protective molecules, indicating that acrolein toxicity may not be mediated via reactive oxygen species. Most strikingly, a mutant lacking an old yellow enzyme (OYE2) was identified as being acrolein sensitive. Old yellow enzymes are known to reduce alpha,beta-unsaturated carbonyl compounds in vitro, but their physiological roles have remained uncertain. We show that mutants lacking OYE2, but not OYE3, are sensitive to acrolein, and overexpression of both isoenzymes increases acrolein tolerance. Our data indicate that OYE2 is required for basal levels of tolerance, whereas OYE3 expression is particularly induced following acrolein stress. Despite the range of alpha,beta-unsaturated carbonyl compounds that have been identified as substrates of old yellow enzymes in vitro, we show that old yellow enzymes specifically mediate resistance to small alpha,beta-unsaturated carbonyl compounds, such as acrolein, in vivo.     

Achievements and perspectives in yeast acetic acid-induced programmed cell death pathways

The use of non-mammalian model organisms, including yeast Saccharomyces cerevisiae, can provide new insights into eukaryotic PCD (programmed cell death) pathways. In the present paper, we report recent achievements in the elucidation of the events leading to PCD that occur as a response to yeast treatment with AA (acetic acid). In particular, ROS (reactive oxygen species) generation, cyt c (cytochrome c) release and mitochondrial function and proteolytic activity will be dealt with as they vary along the AA-PCD time course by using both wild-type and mutant yeast cells. Two AA-PCD pathways are described sharing common features, but distinct from one another with respect to the role of ROS and mitochondria, the former in which YCA1 acts upstream of cyt c release and caspase-like activation in a ROS-dependent manner and the latter in which cyt c release does not occur, but caspase-like activity increases, in a ROS-independent manner.     

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.     

Quasi-programmed aging of budding yeast: a trade-off between programmed processes of cell proliferation,differentiation, stress response,survival and death defines yeast lifespan

Recent findings suggest that evolutionarily distant organisms share the key features of the aging process and exhibit similar mechanisms of its modulation by certain genetic, dietary and pharmacological interventions. The scope of this review is to analyze mechanisms that in the yeast Saccharomyces cerevisiae underlie: (1) the replicative and chronological modes of aging; (2) the convergence of these 2 modes of aging into a single aging process; (3) a programmed differentiation of aging cell communities in liquid media and on solid surfaces; and (4) longevity-defining responses of cells to some chemical compounds released to an ecosystem by other organisms populating it. Based on such analysis, we conclude that all these mechanisms are programs for upholding the long-term survival of the entire yeast population inhabiting an ecological niche; however, none of these mechanisms is a ?program of aging? - i.e., a program for progressing through consecutive steps of the aging process.     

Conserved actin cysteine residues are oxidative stress sensors that can regulate cell death in yeast

Actin's functional complexity makes it a likely target of oxidative stress but also places it in a prime position to coordinate the response to oxidative stress. We have previously shown that the NADPH oxidoreductase Oye2p protects the actin cytoskeleton from oxidative stress. Here we demonstrate that the physiological consequence of actin oxidation is to accelerate cell death in yeast. Loss of Oye2p leads to reactive oxygen species accumulation, activation of the oxidative stress response, nuclear fragmentation and DNA degradation, and premature chronological aging of yeast cells. The oye2Delta phenotype can be completely suppressed by removing the potential for formation of the actin C285-C374 disulfide bond, the likely substrate of the Oye2p enzyme or by treating the cells with the clinically important reductant N-acetylcysteine. Because these two cysteines are coconserved in all actin isoforms, we theorize that we have uncovered a universal mechanism whereby actin helps to coordinate the cellular response to oxidative stress by both sensing and responding to oxidative load.     

Salt stress induces programmed cell death in prokaryotic organism Anabaena

AIMS: Our main interest is to check if programmed cell death (PCD) can occur in prokaryotic algae and if the morphological and biochemical features of PCD are conserved. METHODS AND RESULTS: Using TUNEL labelling, fluorescence and light microscopy and DNA gel electrophoresis, we found that cell death with features similar to those in metazoan PCD could be induced in different Anabaena strains after exposure to univalent-cation salts at moderate concentration. These features included specific DNA fragmentation, cytoplasmic vacuolation, and the progressive disorganization, fragmentation and subsequent autolysis of the cell corpse. Further analyses of cell viability and proteinase activity revealed that increased protease activities, decreased DNA content, and loss of plasmalemma integrity were related to the PCD process. CONCLUSIONS: The results showed that like PCD in eukaryotes, PCD in Anabaena is an active process, and is an adaptation to adverse environments. The features of PCD shared between eukaryotes and Anabaena suggest that PCD mechanisms are conserved during evolution. SIGNIFICANCE AND IMPACT OF THE STUDY: The results will contribute greatly to our understanding of PCD origin and evolution, and are potentially useful in controlling the deluge of algae in some lakes.     

The endoplasmic reticulum stress induced by highly expressed OsrAAT reduces seed size via pre-mature programmed cell death

The high accumulation of a recombinant protein in rice endosperm causes endoplasmic reticulum (ER) stress and in turn dramatically affects endogenous storage protein expression, protein body morphology and seed phenotype. To elucidate the molecular mechanisms underlying these changes in transgenic rice seeds, we analyzed the expression profiles of endogenous storage proteins, ER stress-related and programmed cell death (PCD)-related genes in transgenic lines with different levels of Oryza sativa recombinant alpha antitrypsin (OsrAAT) expression. The results indicated that OsrAAT expression induced the ER stress and that the strength of the ER stress was dependent on OsrAAT expression levels. It in turn induced upregulation of the expression of the ER stress response genes and downregulation of the expression of the endogenous storage protein genes in rice endosperm. Further experiments showed that the ER stress response upregulated the expression of PCD-related genes to disturb the rice endosperm development and induced pre-mature PCD. As consequence, it resulted in decrease of grain weight and size. The mechanisms for the detriment seed phenotype in transgenic lines with high accumulation of the recombinant protein were elucidated.     

Hydrogen peroxide and superoxide anion production during acetic acid-induced yeast programmed cell death

Hydrogen peroxide production in yeast cells undergoing programmed cell death in response to acetic acid occurred in the majority of live cells 15 min after death induction and was no longer detectable after 60 min. Superoxide anion production was found later, 60 and 90 min after death induction when cells viability was 60 and 30%, respectively. In cells protected from death due to acid stress adaptation neither hydrogen peroxide nor superoxide anion could be observed after acetic acid treatment. The early production of hydrogen peroxide in cells in which survival was 100% could play a major role in acetic acid-induced programmed cell death signaling. Superoxide anion is assumed to be generated in cells already en route to acetic acid-induced programmed cell death.     

Cucumber BAX inhibitor-1, a conserved cell death suppressor and a negative programmed cell death regulator under cold stress

Programmed cell death (PCD) is a genetically controlled and conserved process in eukaryotes during development as well as in response to pathogens and other stresses. BAX inhibitor-1 (BI-1) has been implicated as an anti-PCD factor which is highly conserved in plants. Sequence of putative cucumber BI-1 protein exhibited 77.7 % identity and 91.2 % positive value with the homologue Blast BI-1 protein of Arabidopsis thaliana (AtBI-1). This highly homologous protein to the AtBI-1 protein was named CsBI-1. This protein contains an open reading frame (ORF) of 250 amino acids with a BAX inhibitor domain and five transmembrane regions conserved among members of the BI-1 family. Primers designed by the cDNA of CsBI-1gene were used for further sequencing. Cell death in cold-stored cucumber developed concomitantly with increased expression of the CsBI-1 gene and reached maximum at day 6. However, cell death accelerated significantly after 9 d when sharp decrease of the CsBI-1 expression occurred. After warming to 20 °C, expression of the CsBI-1 gene was the highest at day 3, decreased afterwards, and the lowest expression was detected at day 9 when PCD obviously appeared. The overall results indicate that CsBI-1 is cucumber homologue of Arabidopsis thaliana AtBI-1 gene. CsBI-1 is a conserved cell death suppressor induced by cold stress and a negative regulator of PCD.     

Bcl-2 family members inhibit oxidative stress-induced programmed cell death in Saccharomyces cerevisiae

Selected antiapoptotic genes were expressed in baker's yeast (Saccharomyces cerevisiae) to evaluate cytoprotective effects during oxidative stress. When exposed to treatments resulting in the generation of reactive oxygen species (ROS), including H(2)O(2), menadione, or heat shock, wild-type yeast died and exhibited apoptotic-like characteristics, consistent with previous studies. Yeast strains were generated expressing nematode ced-9, human bcl-2, or chicken bcl-xl genes. These transformants tolerated a range of oxidative stresses, did not display features associated with apoptosis, and remained viable under conditions that were lethal to wild-type yeast. Yeast strains expressing a mutant antiapoptotic gene (bcl-2 deltaalpha 5-6), known to be nonfunctional in mammalian cells, were unable to tolerate any of the ROS-generating insults. These data are the first report showing CED-9 has cytoprotective effects against oxidative stress, and add CED-9 to the list of Bcl-2 protein family members that modulate ROS-mediated programmed cell death. In addition, these data indicate that Bcl-2 family members protect wild-type yeast from physiological stresses. Taken together, these data support the concept of the broad evolutionary conservation and functional similarity of the apoptotic processes in eukaryotic organisms.