Victorin induction of an apoptotic/senescence-like response in oats.

Victorin is a host-selective toxin produced by Cochliobolus victoriae, the causal agent of victoria blight of oats. Previously, victorin was shown to be bound specifically by two proteins of the mitochondrial glycine decarboxylase complex, at least one of which binds victorin only in toxin-sensitive genotypes in vivo. This enzyme complex is involved in the photorespiratory cycle and is inhibited by victorin, with an effective concentration for 50% inhibition of 81 pM. The photorespiratory cycle begins with ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), and victorin was found to induce a specific proteolytic cleavage of the Rubisco large subunit (LSU). Leaf slices incubated with victorin for 4 hr in the dark accumulated a form of the LSU that is cleaved after the 14th amino acid. This proteolytic cleavage was prevented by the protease inhibitors E-64 and calpeptin. Another primary symptom of victorin treatment is chlorophyll loss, which along with the specific LSU cleavage is suggestive of a victorin-induced, senescence-like response. DNA from victorin-treated leaf slices showed a pronounced laddering effect, which is typical of apoptosis. Calcium appeared to play a role in mediating the plant response to victorin because LaCl3 gave near-complete protection against victorin, preventing both leaf symptoms and LSU cleavage. The ethylene inhibitors aminooxyacetic acid and silver thiosulfate also gave significant protection against victorin-induced leaf symptoms and prevented LSU cleavage. The symptoms resulting from victorin treatment suggest that victorin causes premature senescence of leaves.     

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.     

The oat mitochondrial permeability transition and its implication in victorin binding and induced cell death

The mitochondrion has emerged as a key regulator of apoptosis, a form of animal programmed cell death (PCD). The mitochondrial permeability transition (MPT), facilitated by a pore-mediated, rapid permeability increase in the inner membrane, has been implicated as an early and critical step of apoptosis. Victorin, the host-selective toxin produced by Cochliobolus victoriae, the causal agent of victoria blight of oats, has been demonstrated to bind to the mitochondrial P-protein and also induces a form of PCD. Previous results suggest that a MPT may facilitate victorin's access to the mitochondrial matrix and binding to the P-protein: (i) victorin-induced cell death displays features similar to apoptosis; (ii) in vivo, victorin binds to the mitochondrial P-protein only in toxin-sensitive genotypes whereas victorin binds equally well to P-protein isolated from toxin-sensitive and insensitive oats; (iii) isolated, untreated mitochondria are impermeable to victorin. The data implicate an in vivo change in mitochondrial permeability in response to victorin. This study focused on whether oat mitochondria can undergo a MPT. Isolated oat mitochondria demonstrated high-amplitude swelling when treated with spermine or Ca2+ in the presence of the Ca2+-ionophore A23187, and when treated with mastoparan, an inducer of the MPT in rat liver mitochondria. In all cases, swelling demonstrated size exclusion in the range 0.9-1.7 kDa, similar to that found in animal mitochondria. Further, MPT-inducing conditions permitted victorin access to the mitochondrial matrix and binding to the P-protein. In vivo, victorin treatment induced the collapse of mitochondrial transmembrane potential within 2 h, indicating a MPT. Also, the victorin-induced collapse of membrane potential was clearly distinct from that induced by uncoupling respiration, as the latter event prevented the victorin-induced PCD response and binding to P-protein. These results demonstrate that a MPT can occur in oat mitochondria in vitro, and are consistent with the hypothesis that an MPT, which allows victorin access to the mitochondrial matrix and binding to the P-protein, occurs in vivo during victorin-induced PCD.     

Involvement of the proteasome in the programmed cell death of NGF-deprived sympathetic neurons.

Sympathetic neurons undergo programmed cell death (PCD) upon deprivation of nerve growth factor (NGF). PCD of neurons is blocked by inhibitors of the interleukin-1beta converting enzyme (ICE)/Ced-3-like cysteine protease, indicating involvement of this class of proteases in the cell death programme. Here we demonstrate that the proteolytic activities of the proteasome are also essential in PCD of neurons. Nanomolar concentrations of several proteasome inhibitors, including the highly selective inhibitor lactacystin, not only prolonged survival of NGF-deprived neurons but also prevented processing of poly(ADP-ribose) polymerase which is known to be cleaved by an ICE/Ced-3 family member during PCD. These results demonstrate that the proteasome is a key regulator of neuronal PCD and that, within this process, it is involved upstream of proteases of the ICE/Ced-3 family. This order of events was confirmed in macrophages where lactacystin inhibited the proteolytic activation of precursor ICE and the subsequent generation of active interleukin-1beta.     

Modification of the proteolytic fragmentation pattern upon oxidation of cysteines from ribulose 1,5-bisphosphate carboxylase/oxygenase

The proteolytic susceptibility of the native CO(2)-fixing photosynthetic enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39, Rubisco) has been shown to increase in vitro after oxidative treatments that affect cysteine thiols. A limited incubation of oxidized (pretreated with the disulfide cystamine) Rubisco from Chlamydomonas reinhardtii with subtilisin or proteinase K generated fragments of molecular mass about 53 kDa (band I in SDS-PAGE) and 47 kDa (band II) derived from the large subunit (55 kDa) of the enzyme. In contrast, proteolysis of the reduced Rubisco (pretreated with the free thiol cysteamine) produced only the 53 kDa band. The same fragmentation pattern was reproduced with Rubiscos from other algae and higher plants, as well as with other chemical modifications of protein cysteines. N-terminal sequencing of the fragments showed that band I arised from clipping the unstructured N-terminal stretch of the large subunit up to Lys18. Band II was generated by a cleavage close to Val69. The increased susceptibility of the oxidized form resulted from proteases gaining access to a loop (from Ser61 to Thr68) located between stretches of secondary structure that form the N-terminal domain. Native electrophoresis and kinetic analysis of fragment accumulation during subtilisin digestion demonstrated that subunit dissociation was induced by the proteolytic processing at the Ser61-Thr68 loop, which is characteristic of the oxidized Rubisco. Holoenzyme dissasembly was readily followed by the full degradation of the released subunits. In contrast, the limited processing to band I observed with the reduced enzyme did not compromise the quaternary structure of the Rubisco hexadecamer, thus preventing further proteolysis.     

Bcl-2 prevents activation of CPP32 cysteine protease and cleavage of poly (ADP-ribose) polymerase and U1-70 kD proteins in staurosporine-mediated apoptosis

Members of the the Bcl-2 and ICE/ced-3 gene families have been implicated as essential components in the control of the cell death pathway. Bcl-2 overexpression can prevent programmed cell death (PCD) in different cell types. ICE/ced-3-like proteases are synthesized as pro-enzymes and are activated by limited proteolysis. When overexpressed in diverse cell types, they trigger PCD. Bcl-2 can inhibit PCD mediated by these proteases, although as yet it is not clear at what specific step in the cell death pathway the protein acts. Here, we demonstrate that CPP32/Yama/Apopain, a member of the ICE/Ced-3 gene family, is processed during staurosporine-induced apoptosis in HeLa cells and that concomitant with CPP32 activation, two other proteins, poly (ADP-ribose) polymerase (PARP) and the U1-70 K small ribonucleoprotein, also undergo proteolysis. Overexpression of Bcl-2 prevents cleavage of CPP32, PARP and U1-70 K and protects HeLa cells from PCD. These results demonstrate that Bcl-2 controls PCD, by acting upstream of CPP32/Yama/Apopain.     

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.     

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.     

Redox modulation of Rubisco conformation and activity through its cysteine residues

Treatment of purified Rubisco with agents that specifically oxidize cysteine-thiol groups causes catalytic inactivation and increased proteolytic sensitivity of the enzyme. It has been suggested that these redox properties may sustain a mechanism of regulating Rubisco activity and turnover during senescence or stress. Current research efforts are addressing the structural basis of the redox modulation of Rubisco and the identification of critical cysteines. Redox shifts result in Rubisco conformational changes as revealed by the alteration of its proteolytic fragmentation pattern upon oxidation. In particular, the augmented susceptibility of Rubisco to proteases is due to increased exposure of a small loop (between Ser61 and Thr68) when oxidized. Progressive oxidation of Rubisco cysteines using disulphide/thiol mixtures at different ratios have shown that inactivation occurs under milder oxidative conditions than proteolytic sensitization, suggesting the involvement of different critical cysteines. Site-directed mutagenesis of conserved cysteines in the Chlamydomonas reinhardtii Rubisco identified Cys449 and Cys459 among those involved in oxidative inactivation, and Cys172 and Cys192 as the specific target for arsenite. The physiological importance of Rubisco redox regulation is supported by the in vivo response of the cysteine mutants to stress conditions. Substitution of Cys172 caused a pronounced delay in stress-induced Rubisco degradation, while the replacement of the functionally redundant Cys449-Cys459 pair resulted in an enhanced catabolism with a faster high-molecular weight polymerization and translocation to membranes. These results suggest that several cysteines contribute to a sequence of conformational changes that trigger the different stages of Rubisco catabolism under increasing oxidative conditions.     

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 VPE-like protease NtTPE8 exclusively expresses in the integumentary tapetum and is involved in seed development

Programmed cell death(PCD) is an essential process for development, and shows conserved cytological features in both plants and animals. Caspases are well-known critical components of the PCD machinery in animals. However, currently few typical counterparts have been identified in plants and only several caspase-like proteases are known to be involved in plant PCD, indicating the existence of great challenge for confirming new caspase-like proteases and elucidating the mechanisms regulating plant PCD. Here, we report a novel cysteine protease, NtTPE8, which was extracted from tobacco seeds and confirmed as a new caspase-like protease.Recombinant NtTPE8 exhibited legumain and caspase-like proteolytic activities, both of which could be inhibited by the pan-caspase inhibitor(Z-VAD-FMK). Notably, NtTPE8 possessed several caspase activities and the capacity to cleave the cathepsin H substrate FVR, indicating a unique character of NtTPE8. NtTPE8 was exclusively expressed in the integumentary tapetum and thus, is the first specific molecular marker reported to date for this cell type. Downregulation of NtTPE8 caused seed abortion, via disturbing early embryogenesis, indicating its critical role in embryogenesis and seed development. In conclusion, we identified a novel caspase-like cysteine protease, NtTPE8,exclusively expressed in the integumentary tapetum that is involved in seed development.     

Current concepts in apoptosis: The physiological suicide program revisited

Apoptosis, or programmed cell death (PCD), involves a complex network of biochemical pathways that normally ensure a homeostatic balance between cellular proliferation and turnover in nearly all tissues. Apoptosis is essential for the body, as its deregulation can lead to several diseases. It plays a major role in a variety of physiological events, including embryonic development, tissue renewal, hormone-induced tissue atrophy, removal of inflammatory cells, and the evolution of granulation tissue into scar tissue. It also has an essential role in wound repair. The various cellular and biochemical mechanisms involved in apoptosis are not fully understood. However, there are two major pathways, the extrinsic pathway (receptor-mediated apoptotic pathway) and the intrinsic pathway (mitochondria-mediated apoptotic pathway), which are both well established. The key component in both is the activation of the caspase cascade. Caspases belong to the family of proteases that ultimately, by cleaving a set of proteins, cause disassembly of the cell. Although the caspase-mediated proteolytic cascade represents a central point in the apoptotic response, its initiation is tightly regulated by a variety of other factors. Among them, Bcl-2 family proteins, TNF and p53 play pivotal roles in the regulation of caspase activation and in the regulation of apoptosis. This review summarizes the established concepts in apoptosis as a physiological cell suicide program, highlighting the recent and significant advances in its study.     

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.     

Gasdermin and Gasdermin-Like Pore-Forming Proteins in Invertebrates,Fungi and Bacteria

The gasdermin family of pore-forming proteins (PFPs) has recently emerged as key molecular players controlling immune-related cell death in mammals. Characterized mammalian gasdermins are activated through proteolytic cleavage by caspases or serine proteases, which remove an inhibitory carboxy-terminal domain, allowing the pore-formation process. Processed gasdermins form transmembrane pores permeabilizing the plasma membrane, which often results in lytic and inflammatory cell death. While the gasdermin-dependent cell death (pyroptosis) has been predominantly characterized in mammals, it now has become clear that gasdermins also control cell death in early vertebrates (teleost fish) and invertebrate animals such as corals (Cnidaria). Moreover, gasdermins and gasdermin-like proteins have been identified and characterized in taxa outside of animals, notably Fungi and Bacteria. Fungal and bacterial gasdermins share many features with mammalian gasdermins including their mode of activation through proteolysis. It has been shown that in some cases the proteolytic activation is executed by evolutionarily related proteases acting downstream of proteins resembling immune receptors controlling pyroptosis in mammals. Overall, these findings establish gasdermins and gasdermin-regulated cell death as an extremely ancient mechanism of cellular suicide and build towards an understanding of the evolution of regulated cell death in the context of immunology. Here, we review the broader gasdermin family, focusing on recent discoveries in invertebrates, fungi and bacteria.     

Differential sensitivity of intestinal brush border enzymes to pancreatic and lysosomal proteases.

Isolated human intestinal brush border membranes were used as sources of enzyme to study their degradation by proteolytic enzymes. Human intestinal brush border hydrolases undergo degradation by two separate proteolytic systems. Sucrase and alkaline phosphatase are degraded by pancreatic proteases (e.g. chymotrypsin) at neutral pH, whereas trehalase is degraded by lysosomal extracts at acid pH. Both the membrane bound and membrane free isolated enzymes had similar sensitivity to proteolytic enzymes. Thus, initial removal from the membrane is not essential as a prerequisite to proteolysis. It is postulated that the brush border membrane of the intestine is subject to proteolysis by pancreatic enzymes from the external cell surface and by lysosomal proteases within the cell.