Caspase-like Activity Research Articles

P30 - Optimization of in vitro measurement of proteasome activity in mammalian cells using fluorogenic substrates

The proteasome is the major multi-catalytic machinery responsible for protein degradation and maintenance of the proteome. The 26S proteasome is an ATP-dependent proteolytic complex, dedicated to the degradation of poly-ubiquitinated proteins. It consists of a 20S proteolytic core and one or two flanking 19S regulatory complexes. The three catalytic subunits harboring chymotrypsin-like (CT-L), trypsin-like (T-L), and caspase-like (C-L; also termed PGPH) activities respectively reside in the 20S proteasome that can also exist in a free form and degrade oxidized and unfolded proteins. Impaired proteasome function has been implicated in the pathogenesis of a number of diseases including Alzheimer’s disease, diabetes, cancer and aging. The emerging interest in proteasome function as diagnostic marker of various human pathologies and therapeutic target necessitates the development of accurate, sensitive and reliable methodologies for the assessment of proteasome activity. Herein, we describe an optimization procedure for the measurement of CT-L, T-L and C-L activities in cell lysates of fibroblasts (HFL-1), melanocytes (B16F10) and peripheral blood mononuclear cells (PBMCs) using fluorogenic peptide substrates in a mid-throughput 96-well plate format. Optimization involves the composition of cell lysis and assay buffers, and the determination of the concentrations of specific fluorogenic substrates and protein content in the reaction to attain appropriate linear catalytic response during measurement. Additional parameters assessed include the concentration of the cell lysate and of ATP in the cell lysis and assay buffers. Our methodological analysis provides useful guidelines for the accurate and rapid determination of proteasome activity in various cell types.

Homogeneous, bioluminescent proteasome assays.

Protein degradation is mediated predominantly through the ubiquitin-proteasome pathway. The importance of the proteasome in regulating degradation of proteins involved in cell-cycle control, apoptosis, and angiogenesis led to the recognition of the proteasome as a therapeutic target for cancer. The proteasome is also essential for degrading misfolded and aberrant proteins, and impaired proteasome function has been implicated in neurodegerative and cardiovascular diseases. Robust, sensitive assays are essential for monitoring proteasome activity and for developing inhibitors of the proteasome. Peptide-conjugated fluorophores are widely used as substrates for monitoring proteasome activity, but fluorogenic substrates can exhibit significant background and can be problematic for screening because of cellular autofluorescence or interference from fluorescent library compounds. Furthermore, fluorescent proteasome assays require column-purified 20S or 26S proteasome (typically obtained from erythrocytes), or proteasome extracts from whole cells, as their samples. To provide assays more amenable to high-throughput screening, we developed a homogeneous, bioluminescent method that combines peptide-conjugated aminoluciferin substrates and a stabilized luciferase. Using substrates for the chymotrypsin-like, trypsin-like, and caspase-like proteasome activities in combination with a selective membrane permeabilization step, we developed single-step, cell-based assays to measure each of the proteasome catalytic activities. The homogeneous method eliminates the need to prepare individual cell extracts as samples and has adequate sensitivity for 96- and 384-well plates. The simple "add and read" format enables sensitive and rapid proteasome assays ideal for inhibitor screening.

Heat shock protein 90 and calcineurin pathway inhibitors enhance the efficacy of triazoles against Scedosporium prolificans via induction of apoptosis.

Scedosporium prolificans is a pathogenic mold resistant to current antifungals, and infection results in high mortality. Simultaneous targeting of both ergosterol biosynthesis and heat shock protein 90 (Hsp90) or the calcineurin pathway in S. prolificans may be an important strategy for enhancing the potency of antifungal agents. We hypothesized that the inactive triazoles posaconazole (PCZ) and itraconazole (ICZ) acquire fungicidal activity when combined with the calcineurin inhibitor tacrolimus (TCR) or Hsp90 inhibitor 17-demethoxy-17-(2-propenylamino) geldanamycin (17AAG). PCZ, ICZ, TCR and 17AAG alone were inactive in vitro against S. prolificans spores (MICs > 128 μg/ml). In contrast, MICs for PCZ or ICZ in combination with TCR or 17AAG (0.125-0.50 μg/ml) were much lower compared with drug alone. In addition PCZ and ICZ in combination with TCR or 17AAG became fungicidal. Because apoptosis is regulated by the calcineurin pathway in fungi and is under the control of Hsp90, we hypothesized that this synergistic fungicidal effect is mediated via apoptosis. This observed fungicidal activity was mediated by increased apoptosis of S. prolificans germlings, as evidenced by reactive oxygen species accumulation, decreased mitochondrial membrane potential, phosphatidylserine externalization, and DNA fragmentation. Furthermore, induction of caspase-like activity was correlated with TCR or 17AAG + PCZ/ICZ-induced cell death. In conclusion, we report for the first time that PCZ or ICZ in combination with TCR or 17AAG renders S. prolificans exquisitely sensitive to PCZ or ICZ via apoptosis. This finding may stimulate the development of new therapeutic strategies for patients infected with this recalcitrant fungus.

Oxidative and proteolytic profiles of the right and left heart in a model of cancer-induced cardiac cachexia

Cardiac cachexia is a syndrome that has received increased attention in recent years. Although an association between proteolysis and cardiac cachexia has been proposed, the direct influence of oxidative stress on the process has not been demonstrated. In the present study, the right (RH) and left (LH) hearts (atrium and ventricle of each side of the heart) were collected from rats at the 5th and 10th days after phosphate buffer (control) orWalker-256 solid tumour implantation. Immediately after sacrifice, cachexia was determined in tumour-bearing animals by the formula: [(inicial body weight−final body weight+tumour weight+weight gain of control group)/(initial body weight+body mass gain of control group)]×100%; RH and LH were stored until use. Oxidative stress and proteolysis were determined in each collected sample. In addition, heart samples were collected from a separate set of animals to determine the thickness of the left and right ventricles. Cachexia values increased over time after tumour implantation from 6.85% at the 5th day to 17.76% at the 10th day. There was no significant difference in LH wet weight and ventricle thickness compared with the control, where as RH wet weight (0.109±0.09g at the 5th day and 0.093±0.09g at the 10th day) and thickness (420±16μm at the 5th day and 279±08μm at the 10th day) were significantly decreased at both time points when compared with control values (0.153±0.06g and 607±21μm, respectively). tert-Butyl-stimulated chemiluminescence analysis revealed a significant increase in the LH and decrease in the RH oxidative stress profiles. Carbonylated proteins increased in the LH (140%, p<0.05) and RH (100%, p<0.05) at the 5th day, and significantly decreased in both sides on the 10th day compared to controls. Chemotrypsin-like, caspase-like, and calpain-like activities were evaluated by chemiluminescence, and only calpain-like activity was found to increase at the 5th day in the RH. In the LH, all proteolytic activities systems were decreased when compared with controls. Together, these results demonstrate that oxidative stress appears to play a different role in mass modulation on the LH and RH. The proteolytic systems evaluated herein also appear to have different effects on the responses developed during cardiac cachexia in the two sides of the heart.

A novel copper(I) complex induces ER-stress-mediated apoptosis and sensitizes B-acute lymphoblastic leukemia cells to chemotherapeutic agents.

A phosphine copper(I) complex [Cu(thp)4][PF6] (CP) was recently identified as an efficient in vitro antitumor agent. In this study, we evaluated the antiproliferative activity of CP in leukemia cell lines finding a significant efficacy, especially against SEM and RS4;11 cells. Immunoblot analysis showed the activation of caspase-12 and caspase-9 and of the two effector caspase-3 and -7, suggesting that cell death occurred in a caspase-dependent manner. Interestingly we did not observe mitochondrial involvement in the process of cell death. Measures on semipurified proteasome from RS4;11 and SEM cell extracts demonstrated that chymotrypsin-, trypsin- and caspase-like activity decreased in the presence of CP. Moreover, we found an accumulation of ubiquitinated proteins and a remarkable increase of ER stress markers: GRP78, CHOP, and the spliced form of XBP1. Accordingly, the protein synthesis inhibitor cycloheximide significantly protected cancer cells from CP-induced cell death, suggesting that protein synthesis machinery was involved. In well agreement with results obtained on stabilized cell lines, CP induced ER-stress and apoptosis also in primary cells from B-acute lymphoblastic leukemia patients. Importantly, we showed that the combination of CP with some chemotherapeutic drugs displayed a good synergy that strongly affected the survival of both RS4;11 and SEM cells.

Reactive oxygen species burst induced by aluminum stress triggers mitochondria-dependent programmed cell death in peanut root tip cells

Recent studies had certified that aluminum (Al) induced ROS production and programmed cell death (PCD) in higher plants. The relationship between ROS production and PCD occurrence under Al stress is uncovered. The results showed that root elongation inhibition and PCD occurrence was induced by 100 μM AlCl3. Al stress induced ROS burst, up-regulated Rboh and COX gene expression, increased mitochondrial permeability transition pore (MPTP) opening, decreased inner mitochondrial membrane potential (ΔΨm), released cytochrome c from mitochondria to cytoplasm, activated caspase 3-like protease activity. Exogenous H2O2 aggravated the changes caused by Al and accelerated PCD occurrence, but ROS scavenger CAT and AsA reversed the changes caused by Al and inhibited PCD production. A potential cascade of cellular events during Al induced PCD via mitochondria dependent pathway and the mechanism of ROS on regulating PCD induced by Al is proposed.

Computational Inhibition Studies of the Human Proteasome by Argyrin‐Based Analogues with Subunit Specificity

A computational procedure was developed to study the subunit-specific interactions of the proteasome inhibitors argyrin A and F, with the aim of indentifying the determinants of subunit selectivity. Three-dimensional models of humanized proteasome active sites β1 , β2 and β5 were developed and subsequently used in molecular docking simulations with the argyrin analogues. The subunit selectivity exhibited by each analogue could be explained based on the site-specific interactions and a probability-based specificity parameter derived in this study. A rational approach that involved maximizing site-specific interactions was followed to guide the design of new argyrin analogues as specific inhibitors of the caspase-like (β1 site) activity.

Programmed cell death in kiwifruit stigmatic arms and its relationship to the effective pollination period and the progamic phase

Kiwifruit is a crop with a highly successful reproductive performance, which is impaired by the short effective pollination period of female flowers. This study investigates whether the degenerative processes observed in both pollinated and non-pollinated flowers after anthesis may be considered to be programmed cell death (PCD). Features of PCD in kiwifruit, Actinidia chinensis var. deliciosa, were studied in both non-pollinated and pollinated stigmatic arms using transmission electron microscopy, DAPI (4',6-diamidino-2-phenylindole) staining, TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling) assays, DNA gel electrophoresis and caspase-like activity assays. In the secretory tissues of the stigmatic arms, cell organelles disintegrated sequentially while progressive vacuolization was detected. At the same time, chromatin condensation, nuclear deformation, and DNA fragmentation and degradation were observed. These features were detected in both non-pollinated and pollinated stigmatic arms; they were evident in the stigmas of pollinated flowers by the second day after anthesis but only by 4 d after anthesis in non-pollinated flowers. In addition, in pollinated stigmatic arms, these features were first initiated in the stigma and gradually progressed through the style, consistent with pollen tube growth. This timing of events was also observed in both non-pollinated and pollinated stigmatic arms for caspase-3-like activity. The data provide evidence to support the hypothesis that PCD processes occurring in the secretory tissue of non-pollinated kiwifruit stigmatic arms could be the origin for the observed short effective pollination period. The results obtained in the secretory tissue of pollinated kiwifruit stigmatic arms upon pollination support the idea that PCD might be accelerated by pollination, pointing to the involvement of PCD during the progamic phase.

Effects of a novel proteasome inhibitor BU-32 on multiple myeloma cells.

Proteasome inhibition is associated with substantial antitumor effects in preclinical models of multiple myeloma (MM) as well as in patients. However, results of recent clinical trials to evaluate the effect of the proteasome inhibitor Bortezomib (Velcade(®), also called PS-341) in MM patients have shown limited activity when used as a single agent. This underscores the need to find new efficacious and less toxic proteasome inhibitors. Recently, carfilzomib was approved for the treatment of refractory/relapsed MM and several new agents have been introduced into the clinic, including marizomib and MLN9708, and trials investigating these second-generation proteasome inhibitors have demonstrated promising results. We have recently synthesized a novel proteasome inhibitor, BU-32, and tested its growth inhibitory effects in different human MM cells including RPMI8226, MM.1S, MM.1R, and U266. In this study, we evaluate the efficacy of the novel proteasome inhibitor BU-32 (NSC D750499) using an in vitro MM model. BU-32 exhibits strong cytotoxicity in a panel of MM cell lines--RPMI8226, MM1S, MM1R, and U266. In addition, we demonstrate by proteasome inhibition assay that BU-32 potently inhibits the chymotryptic- and caspase-like activities of the 26S proteasome. We further show from Annexin V-FITC binding studies that BU-32, like Bortezomib, induces apoptosis in a panel of MM cell lines but the effect is more pronounced with BU-32-treated cells. Invasion assay with the MM.1S cell line indicates that BU-32 inhibits the invasiveness of myeloma cells. Results from our studies using real-time PCR array analyses show that BU-32 effectively downregulates an array of angiogenesis and inflammatory markers. Our results suggest that BU-32 might be a potential chemotherapeutic agent with promising antitumor activity for the treatment of MM.

Investigating the cell death mechanism(s) of 4‐hydroxy‐tamoxifen in glioblastoma (411.1)

Tamoxifen (TMX) is widely used as a chemotherapeutic agent in the treatment of estrogen receptor (ER)‐positive breast cancer. Recent studies have demonstrated that TMX inhibits the growth of malignant peripheral nerve sheath tumors (MPNSTs) through an ER‐independent mechanism associated with the induction of autophagy. We hypothesize that TMX could induce autophagic cell death in glioblastoma (GBM), the most common and aggressive malignant astrocytic glioma. In an established human GBM cell line and xenografts, TMX caused a concentration‐dependent decrease in cell viability that was not mediated by caspase 3‐like enzymatic activity. We observed a concentration‐dependent decrease in KRAS protein levels and an accumulation of LC3‐II, a surrogate marker of autophagic vacuoles. Finally, because epidermal growth factor receptor (EGFR) amplification is frequently associated with GBM and elevated EGFR signaling is known to contribute to the malignant properties of GBM, we examined the effects of TMX on EGFR and found a decrease in EGFR protein and Akt signaling. These data suggest that TMX induces GBM cell death through a caspase‐independent, autophagy‐related mechanism and should be considered as a potential therapeutic agent in GBMs and other malignancies that are not amenable to apoptotic‐targeting therapies.Grant Funding Source: NINDS

The Membrane-Associated Transcription Factor NAC089 Controls ER-Stress-Induced Programmed Cell Death in Plants

The unfolded protein response (UPR) is activated to sustain cell survival by reducing misfolded protein accumulation in the endoplasmic reticulum (ER). The UPR also promotes programmed cell death (PCD) when the ER stress is severe; however, the underlying molecular mechanisms are less understood, especially in plants. Previously, two membrane-associated transcriptions factors (MTFs), bZIP28 and bZIP60, were identified as the key regulators for cell survival in the plant ER stress response. Here, we report the identification of another MTF, NAC089, as an important PCD regulator in Arabidopsis (Arabidopsis thaliana) plants. NAC089 relocates from the ER membrane to the nucleus under ER stress conditions. Inducible expression of a truncated form of NAC089, in which the transmembrane domain is deleted, induces PCD with increased caspase 3/7-like activity and DNA fragmentation. Knock-down NAC089 in Arabidopsis confers ER stress tolerance and impairs ER-stress-induced caspase-like activity. Transcriptional regulation analysis and ChIP-qPCR reveal that NAC089 plays important role in regulating downstream genes involved in PCD, such as NAC094, MC5 and BAG6. Furthermore, NAC089 is up-regulated by ER stress, which is directly controlled by bZIP28 and bZIP60. These results show that nuclear relocation of NAC089 promotes ER-stress-induced PCD, and both pro-survival and pro-death signals are elicited by bZIP28 and bZIP60 during plant ER stress response.

Self-incompatibility in Papaver: advances in integrating the signalling network

Self-fertilization, which results in reduced fitness of offspring, is a common problem in hermaphrodite angiosperms. To prevent this, many plants utilize SI (self-incompatibility), which is determined by the multi-allelic S-locus, that allows discrimination between self (incompatible) and non-self (compatible) pollen by the pistil. In poppy (Papaver rhoeas), the pistil S-determinant (PrsS) is a small secreted protein which interacts with the pollen S-determinant PrpS, a ~20 kDa novel transmembrane protein. Interaction of matching pollen and pistil S-determinants results in self-recognition, initiating a Ca²⁺-dependent signalling network in incompatible pollen. This triggers several downstream events, including alterations to the cytoskeleton, phosphorylation of sPPases (soluble inorganic pyrophosphatases) and an MAPK (mitogen-activated protein kinase), increases in ROS (reactive oxygen species) and nitric oxide (NO), and activation of several caspase-like activities. This results in the inhibition of pollen tube growth, prevention of self-fertilization and ultimately PCD (programmed cell death) in incompatible pollen. The present review focuses on our current understanding of the integration of these signals with their targets in the SI/PCD network. We also discuss our recent functional expression of PrpS in Arabidopsis thaliana pollen.

PGPR regulate caspase-like activity, programmed cell death, and antioxidant enzyme activity in paddy under salinity.

The response of two root associated bacteria Pseudomonas pseudoalcaligenes and Bacillus pumilus were studied in the (salt-sensitive) rice GJ17 cultivar to salinity under controlled environmental growth conditions for protection of plant from adverse effect of salinity. Salinity affects the growth of salt-sensitive cultivar, but inoculation of plant growth promoting rhizobacteria (PGPR) reduces the harmful effect of salinity. The present study states that PGPR helps to reduce lipid peroxidation and superoxide dismutase activity in salt-sensitive GJ17 cultivar under salinity and play an important role in the growth regulation for positive adaptation of plants to salt stress. This study shows that inoculation of paddy (Oryza sativa) with such bacteria could provide salt-tolerant ability by reducing the toxicity of reactive oxygen species by reducing plant cell membrane index, cell caspase-like protease activity, and programmed cell death and hence resulted in increase cell viability. As these isolates remain associated with the roots, the effects of tolerance against salinity are observed here. Results also indicate that isolated PGPR strain help in alleviating up to 1.5% salinity stress as well as improve tolerance.

Inhibition of Human and Yeast 20S Proteasome by Analogues of Trypsin Inhibitor SFTI-1

Starting from the primary structure of sunflower trypsin inhibitor SFTI-1, we designed novel non-covalent inhibitors of human and yeast 20S proteasomes. Peptides with Arg residue in P1 position and two basic amino acid residues (Lys or/and Arg) in P2′ and P3′ positions strongly inhibited chymotrypsin-like and caspase-like activities, while trypsin-like activity was poorly modified. We found that some SFTI-1 analogues up-regulated exclusively the chymotrypsin-like activity of latent yeast 20S proteasome.

Interactive effects of CO2 and trace metals on the proteasome activity and cellular stress response of marine bivalves Crassostrea virginica and Mercenaria mercenaria

Increased anthropogenic emission of CO2 changes the carbonate chemistry and decreases the pH of the ocean. This can affect the speciation and the bioavailability of metals in polluted habitats such as estuaries. However, the effects of acidification on metal accumulation and stress response in estuarine organisms including bivalves are poorly understood. We studied the interactive effects of CO2 and two common metal pollutants, copper (Cu) and cadmium (Cd), on metal accumulation, intracellular ATP/ubiquitin-dependent protein degradation, stress response and energy metabolism in two common estuarine bivalves—Crassostrea virginica (eastern oyster) and Mercenaria mercenaria (hard shell clam). Bivalves were exposed for 4–5 weeks to clean seawater (control) and to either 50μgL−1 Cu or 50μgL−1 Cd at one of three partial pressures of CO2 (PCO2 ∼395, ∼800 and ∼1500μatm) representative of the present-day conditions and projections of the Intergovernmental Panel for Climate Change (IPCC) for the years 2100 and 2250, respectively. Clams accumulated lower metal burdens than oysters, and elevated PCO2 enhanced the Cd and Cu accumulation in mantle tissues in both species. Higher Cd and Cu burdens were associated with elevated mRNA expression of metal binding proteins metallothionein and ferritin. In the absence of added metals, proteasome activities of clams and oysters were robust to elevated PCO2, but PCO2 modulated the proteasome response to metals. Cd exposure stimulated the chymotrypsin-like activity of the oyster proteasome at all CO2 levels. In contrast, trypsin- and caspase-like activities of the oyster proteasome were slightly inhibited by Cd exposure in normocapnia but this inhibition was reversed at elevated PCO2. Cu exposure inhibited the chymotrypsin-like activity of the oyster proteasome regardless of the exposure PCO2. The effects of metal exposure on the proteasome activity were less pronounced in clams, likely due to the lower metal accumulation. However, the general trends (i.e. an increase during Cd exposure, inhibition during exposure to Cu, and overall stimulatory effects of elevated PCO2) were similar to those found in oysters. Levels of mRNA for ubiquitin and tumor suppressor p53 were suppressed by metal exposures in normocapnia in both species but this effect was alleviated or reversed at elevated PCO2. Cellular energy status of oysters was maintained at all metal and CO2 exposures, while in clams the simultaneous exposure to Cu and moderate hypercapnia (∼800μatm PCO2) led to a decline in glycogen, ATP and ADP levels and an increase in AMP indicating energy deficiency. These data suggest that environmental CO2 levels can modulate accumulation and physiological effects of metals in bivalves in a species-specific manner which can affect their fitness and survival during the global change in estuaries.

Proteasome inhibitors in progressive renal diseases

Proteasome (PS) is a sophisticated protein degradation machinery comprising a 20S proteolytic core particle provided with caspase-like, trypsin-like and chymotrypsin-like activities on ubiquitinilated proteins. The products of this selective, complex, controlled and strictly coordinated system play a crucial role in cell cycle progression and apoptosis; activation of transcription factors, cytokines and chemokines; degradation and generation of MHC class I-presented peptides. PS has recently emerged as a promising drug target in cancer therapy, and bortezomib has been approved for refractory multiple myeloma. PS proteolysis is crucial for the degradation of the inhibitory protein IkB of nuclear factor kB (NF-kB), and hence, an interesting field of research has been developed on possible benefits of drugs with anti-PS activity in disease conditions with hyper-expression of NF-kB. PS inhibitors are being adopted in pilot studies in antibody-mediated renal rejection and in AL amyloidosis, with increasing scientific interest in possible applications in lupus, IgA nephropathy, idiopathic nephrotic syndrome and renal fibrosis. The most often used PS inhibitor, bortezomib, has a severe peripheral neurotoxicity, and the search for effective and less toxic PS-targeted drugs is a challenging area also in nephrology.

Cell-line-specific high background in the Proteasome-Glo assay of proteasome trypsin-like activity

Proteasome-Glo is a homogeneous cell-based assay of proteasomal chymotrypsin-like, trypsin-like, and caspase-like activities using luminogenic substrates, commercially available from Promega. Here we report that the background activity from cleavage of the substrate of the trypsin-like sites by nonproteasomal proteases in multiple breast and lung cancer cell lines exceeds the activity of the proteasome. We also observed substantial background chymotrypsin-like activity in some cell lines. Thus, Proteasome-Glo assay must be used with caution, and it is necessary to include a specific proteasome inhibitor to determine the background for each proteasome activity.

Effects of an Anticarcinogenic Bowman-Birk Protease Inhibitor on Purified 20S Proteasome and MCF-7 Breast Cancer Cells

Proteasome inhibitors have been described as an important target for cancer therapy due to their potential to regulate the ubiquitin-proteasome system in the degradation pathway of cellular proteins. Here, we reported the effects of a Bowman-Birk-type protease inhibitor, the Black-eyed pea Trypsin/Chymotrypsin Inhibitor (BTCI), on proteasome 20S in MCF-7 breast cancer cells and on catalytic activity of the purified 20S proteasome from horse erythrocytes, as well as the structural analysis of the BTCI-20S proteasome complex. In vitro experiments and confocal microscopy showed that BTCI readily crosses the membrane of the breast cancer cells and co-localizes with the proteasome in cytoplasm and mainly in nucleus. Indeed, as indicated by dynamic light scattering, BTCI and 20S proteasome form a stable complex at temperatures up to 55°C and at neutral and alkaline pHs. In complexed form, BTCI strongly inhibits the proteolytic chymotrypsin-, trypsin- and caspase-like activities of 20S proteasome, indicated by inhibition constants of 10−7 M magnitude order. Besides other mechanisms, this feature can be associated with previously reported cytostatic and cytotoxic effects of BTCI in MCF-7 breast cancer cells by means of apoptosis.

Endoplasmic reticulum stress-induced PCD and caspase-like activities involved

Plant cells, like cells from other kingdoms, have the ability to self-destruct in a genetically controlled manner. This process is defined as Programmed cell death (PCD). PCD can be triggered by various stimuli in plants including by endoplasmic reticulum (ER) stress. Research in the past two decades discovered that disruption of protein homeostasis in the ER could cause ER stress, which when prolonged/unresolved leads cells into PCD. ER stress-induced PCD is part of several plant processes, for instance, drought and heat stress have been found to elicit ER stress-induced PCD. Despite the importance of ER stress-induced PCD in plants, its regulation remains largely unknown, when compared with its counterpart in animal cells. In mammalian cells, several pro-apoptotic proteases called caspases were found to play a crucial role in ER stress-induced PCD. Over the past decade, several key proteases with caspase-like enzymatic activity have been discovered in plants and implicated in PCD regulation. This review covers what is known about caspase-like enzymatic activities during plant ER stress-induced PCD and discusses possible regulation pathways leading to the activation of relevant proteases in plants.

Proteasome Assay in Cell Lysates.

The ubiquitin-proteasome system (UPS) mediates the majority of the proteolysis seen in the cytoplasm and nucleus of mammalian cells. As such it plays an important role in the regulation of a variety of physiological and pathophysiological processes including tumorigenesis, inflammation and cell death (Ciechanover, 2005; Kisselev and Goldberg, 2001). A number of recent studies have shown that proteasome activity is decreased in a variety of neurological disorders including Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis and stroke as well as during normal aging (Chung et al., 2001; Ciechanover and Brundin, 2003; Betarbet et al., 2005). This decrease in proteasome activity is thought to play a critical role in the accumulation of abnormal and oxidized proteins. Protein clearance by the UPS involves two sequential reactions. The first is the tagging of protein lysine residues with ubiquitin (Ub) and the second is the subsequent degradation of the tagged proteins by the proteasome. We herein describe an assay for the second of these two reactions (Valera et al., 2013). This assay uses fluorogenic substrates for each of the three activities of the proteasome: chymotrypsin-like activity, trypsin-like activity and caspase-like activity. Cleavage of the fluorophore from the substrate by the proteasome results in fluorescence that can be detected with a fluorescent plate reader.