Two proteases with caspase-3-like activity, cathepsin B and proteasome, antagonistically control ER-stress-induced programmed cell death in Arabidopsis.
ABSTRACT: Programmed cell death (PCD) induced by endoplasmic reticulum (ER) stress is implicated in various plant physiological processes, yet its mechanism is still elusive. An activation of caspase-3-like enzymatic activity was clearly demonstrated but the role of the two known plant proteases with caspase-3-like activity, cathepsin B and proteasome subunit PBA1, remains to be established. Both genetic downregulation and chemical inhibition were used to investigate the function of cathepsin B and PBA1 in ER-stress-induced PCD (ERSID). Transcript level and activity labelling of cathepsin B were used to assess activation. To study tonoplast rupture, a plant PCD feature, both confocal and electronic microscopies were used. Cathepsin B downregulation reduced reactive oxygen species (ROS) accumulation and ERSID without affecting the induction of the unfolded protein response (UPR), but downregulation of PBA1 increased UPR and ERSID. Tonoplast rupture was not altered in the cathepsin B mutant and cathepsin B activation was independent of vacuolar processing enzyme (VPE). VPE activity was independent of cathepsin B. ERSID is regulated positively by cathepsin B and negatively by PBA1, revealing a complex picture behind caspase-3-like activity in plants. Cathepsin B may execute its function after tonoplast rupture and works in parallel with VPE.
Project description:BACKGROUND:Vacuolar processing enzymes (VPEs) have been identified as the enzymes that regulate vacuole-mediated programmed cell death (PCD) in plants. The mechanism that VPE regulates the PCD in rice aleurone layers remains unknown. RESULTS:The aleurone layers treated with distilled water exerted caspase-1 and VPE activity, both of which were inhibited by the caspase-1 specific inhibitor Ac-YVAD-CMK but not by the caspase-3 specific inhibitor Ac-DEVD-CHO. However, the caspase-1 and caspase-3 inhibitors weakened the activity of caspase-3. Combined with the effects of endogenous gibberellin (GA) on the induction of OsVPEs, we suggest that the OsVPE3 in the aleurone layers, which exhibits caspase-1-like activity, is a key molecule in GA-induced PCD via regulating the protease with caspase-3-like activity. Many studies have confirmed that vacuolar fusion is an important feature of vacuole-mediated PCD in plants. In this experiment, the process of vacuole fusion was accompanied by changes in the structure of actin filaments (AFs), specifically, their depolymerization and polymerization. The process of vacuolar fusion was accelerated or delayed by the promotion or inhibition of the depolymerization of AFs, respectively. Here, the inhibition of OsVPE3 blocked the depolymerization of AFs and delayed the fusion of vacuoles, indicating that OsVPE3 can regulate the fusion of vacuoles in rice aleurone layers via mediating AFs. Furthermore, the depolymerization of AFs contributed to the up-regulation of OsVPE3 gene expression and VPE activity, resulting in accelerated PCD in rice aleurone layers. However, the inhibitor of VPE reversed the effects of AF depolymerization on the activity of VPE, then postponing the process of PCD, implying that AF can involve in GA-induced PCD of rice aleurone layers by mediating OsVPE3. CONCLUSIONS:Together, activation of OsVPE3 and depolymerization of AFs shortened the process of vacuolation and PCD in rice aleurone layers, and OsVPE3 interacted with AFs during regulation.
Project description:Vacuole fusion is a necessary process for the establishment of a large central vacuole, which is the central location of various hydrolytic enzymes and other factors involved in death at the beginning of plant programmed cell death (PCD). In our report, the fusion of vacuoles has been presented in two ways: i) small vacuoles coalesce to form larger vacuoles through membrane fusion, and ii) larger vacuoles combine with small vacuoles when small vacuoles embed into larger vacuoles. Regardless of how fusion occurs, a large central vacuole is formed in rice (Oryza sativa) aleurone cells. Along with the development of vacuolation, the rupture of the large central vacuole leads to the loss of the intact plasma membrane and the degradation of the nucleus, resulting in cell death. Stabilizing or disrupting the structure of actin filaments (AFs) inhibits or promotes the fusion of vacuoles, which delays or induces PCD. In addition, the inhibitors of the vacuolar processing enzyme (VPE) and cathepsin B (CathB) block the occurrence of the large central vacuole and delay the progression of PCD in rice aleurone layers. Overall, our findings provide further evidence for the rupture of the large central vacuole triggering the PCD in aleruone layers.
Project description:Prolonged endoplasmic reticulum and osmotic stress synergistically activate the stress-induced N-rich protein-mediated signaling that transduces a cell death signal by inducing GmNAC81 (GmNAC6) in soybean. To identify novel regulators of the stress-induced programmed cell death (PCD) response, we screened a two-hybrid library for partners of GmNAC81. We discovered another member of the NAC (NAM-ATAF1,2-CUC2) family, GmNAC30, which binds to GmNAC81 in the nucleus of plant cells to coordinately regulate common target promoters that harbor the core cis-regulatory element TGTG[TGC]. We found that GmNAC81 and GmNAC30 can function either as transcriptional repressors or activators and cooperate to enhance the transcriptional regulation of common target promoters, suggesting that heterodimerization may be required for the full regulation of gene expression. Accordingly, GmNAC81 and GmNAC30 display overlapping expression profiles in response to multiple environmental and developmental stimuli. Consistent with a role in PCD, GmNAC81 and GmNAC30 bind in vivo to and transactivate hydrolytic enzyme promoters in soybean protoplasts. A GmNAC81/GmNAC30 binding site is located in the promoter of the caspase-1-like vacuolar processing enzyme (VPE) gene, which is involved in PCD in plants. We demonstrated that the expression of GmNAC81 and GmNAC30 fully transactivates the VPE gene in soybean protoplasts and that this transactivation was associated with an increase in caspase-1-like activity. Collectively, our results indicate that the stress-induced GmNAC30 cooperates with GmNAC81 to activate PCD through the induction of the cell death executioner VPE.
Project description:Programmed cell death (PCD) is used by plants for development and survival to biotic and abiotic stresses. The role of caspases in PCD is well established in animal cells. Over the past 15 years, the importance of caspase-3-like enzymatic activity for plant PCD completion has been widely documented despite the absence of caspase orthologues. In particular, caspase-3 inhibitors blocked nearly all plant PCD tested. Here, we affinity-purified a plant caspase-3-like activity using a biotin-labelled caspase-3 inhibitor and identified Arabidopsis thaliana cathepsin B3 (AtCathB3) by liquid chromatography with tandem mass spectrometry (LC-MS/MS). Consistent with this, recombinant AtCathB3 was found to have caspase-3-like activity and to be inhibited by caspase-3 inhibitors. AtCathepsin B triple-mutant lines showed reduced caspase-3-like enzymatic activity and reduced labelling with activity-based caspase-3 probes. Importantly, AtCathepsin B triple mutants showed a strong reduction in the PCD induced by ultraviolet (UV), oxidative stress (H2O2, methyl viologen) or endoplasmic reticulum stress. Our observations contribute to explain why caspase-3 inhibitors inhibit plant PCD and provide new tools to further plant PCD research. The fact that cathepsin B does regulate PCD in both animal and plant cells suggests that this protease may be part of an ancestral PCD pathway pre-existing the plant/animal divergence that needs further characterisation.
Project description:BACKGROUND: Developmentally regulated programmed cell death (PCD) is the controlled death of cells that occurs throughout the life cycle of both plants and animals. The lace plant (Aponogeton madagascariensis) forms perforations between longitudinal and transverse veins in spaces known as areoles, via developmental PCD; cell death begins in the center of these areoles and develops towards the margin, creating a gradient of PCD. This gradient was examined using both long- and short-term live cell imaging, in addition to histochemical staining, in order to establish the order of cellular events that occur during PCD. RESULTS: The first visible change observed was the reduction in anthocyanin pigmentation, followed by initial chloroplast changes and the bundling of actin microfilaments. At this stage, an increased number of transvacuolar strands (TVS) was evident. Perhaps concurrently with this, increased numbers of vesicles, small mitochondrial aggregates, and perinuclear accumulation of both chloroplasts and mitochondria were observed. The invagination of the tonoplast membrane and the presence of vesicles, both containing organelle materials, suggested evidence for both micro- and macro-autophagy, respectively. Mitochondrial aggregates, as well as individual chloroplasts were subsequently seen undergoing Brownian motion in the vacuole. Following these changes, fragmentation of nuclear DNA, breakdown of actin microfilaments and early cell wall changes were detected. The vacuole then swelled, causing nuclear displacement towards the plasma membrane (PM) and tonoplast rupture followed closely, indicating mega-autophagy. Subsequent to tonoplast rupture, cessation of Brownian motion occurred, as well as the loss of mitochondrial membrane potential (??m), nuclear shrinkage and PM collapse. Timing from tonoplast rupture to PM collapse was approximately 20 minutes. The entire process from initial chlorophyll reduction to PM collapse took approximately 48 hours. Approximately six hours following PM collapse, cell wall disappearance began and was nearly complete within 24 hours. CONCLUSION: Results showed that a consistent sequence of events occurred during the remodelling of lace plant leaves, which provides an excellent system to study developmental PCD in vivo. These findings can be used to compare and contrast with other developmental PCD examples in plants.
Project description:Vacuolar processing enzymes (VPEs) have received considerable attention due to their caspase-1-like activity and ability to regulate programmed cell death (PCD), which plays an essential role in the development of stenospermocarpic seedless grapes ovules. To characterize VPEs and the relationship between stenospermocarpic grapes and the VPE gene family, we identified 3 Vitis vinifera VPE genes (Vv?VPE, Vv?VPE, and Vv?VPE) from the PN40024 grape genome and cloned the full-length complementary DNAs (cDNAs) from the 'Vitis vinifera cv. Pinot Noir' and 'Vitis vinifera cv. Thompson Seedless' varietals. Each of the VPEs contained a typical catalytic dyad [His (177), Cys (219)] and substrate binding pocket [Arg (112), Arg (389), Ser (395)], except that Ser (395) in the Vv?VPE protein sequence was replaced with alanine. Phylogenetic analysis of 4 Arabidopsis thaliana and 6 Vitis vinifera VPEs revealed that the 10 VPEs form 3 major branches. Furthermore, the 6 grapevine VPEs share a similar gene structure, with 9 exons and 8 introns. The 6 grapevine VPEs are located on 3 different chromosomes. We also tested the enzymatic activity of recombinant VPEs expressed in the Pichia Pastoris expression system and found that the VvVPEs exhibit cysteine peptidase activity. Tissue-specific expression analysis showed that Vv?VPE is only expressed in flowers, buds and ovules, that Vv?VPE is expressed in various tissues, and that Vv?VPE was expressed in roots, flowers, buds and ovules. The results of quantitative real-time PCR (qRT-PCR) suggested that Vv?VPE in seeded grapes increased significantly at 30 days after full-bloom (DAF), close to the timing of endosperm abortion at 32 DAF. These results suggested that Vv?VPE is related to ovule abortion in seedless grapes. Our experiments provide a new perspective for understanding the mechanism of stenospermocarpic seedlessness and represent a useful reference for the further study of VPEs.
Project description:BACKGROUND:Since their discovery, vacuolar processing enzymes (VPEs) have consistently been investigated as programmed cell death (PCD) initiators and participants in plant development and responses to biotic or abiotic stresses, in part due to similarities with the apoptosis regulator caspase-1. However, recent studies show additional functions of VPE in tomatoes, specifically in sucrose accumulation and fruit ripening. RESULTS:Herein, we evaluated the functions of VPE from sweetpotato, initially in expression pattern analyses of IbVPE1 during development and senescence. Subsequently, we identified physiological functions by overexpressing IbVPE1 in Arabidopsis thaliana, and showed reduced leaf sizes and numbers and early flowering, and elucidated the underlying molecular mechanisms. CONCLUSIONS:The present data demonstrate functions of the VPE gene family in development and senescence and in regulation of flowering times, leaf sizes and numbers, and senescence phenotypes in Arabidopsis thaliana.
Project description: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.
Project description:BACKGROUND:Glioma is the most commonly diagnosed primary brain tumor and is characterized by invasive and infiltrative behavior. uPAR and cathepsin B are known to be overexpressed in high-grade gliomas and are strongly correlated with invasive cancer phenotypes. METHODOLOGY/PRINCIPAL FINDINGS:In the present study, we observed that simultaneous downregulation of uPAR and cathepsin B induces upregulation of some pro-apoptotic genes and suppression of anti-apoptotic genes in human glioma cells. uPAR and cathepsin B (pCU)-downregulated cells exhibited decreases in the Bcl-2/Bax ratio and initiated the collapse of mitochondrial membrane potential. We also observed that the broad caspase inhibitor, Z-Asp-2, 6-dichlorobenzoylmethylketone rescued pCU-induced apoptosis in U251 cells but not in 5310 cells. Immunoblot analysis of caspase-9 immunoprecipitates for Apaf-1 showed that uPAR and cathepsin B knockdown activated apoptosome complex formation in U251 cells. Downregulation of uPAR and cathepsin B also retarded nuclear translocation and interfered with DNA binding activity of CREB in both U251 and 5310 cells. Further western blotting analysis demonstrated that downregulation of uPAR and cathepsin B significantly decreased expression of the signaling molecules p-PDGFR-?, p-PI3K and p-Akt. An increase in the number of TUNEL-positive cells, increased Bax expression, and decreased Bcl-2 expression in nude mice brain tumor sections and brain tissue lysates confirm our in vitro results. CONCLUSIONS/SIGNIFICANCE:In conclusion, RNAi-mediated downregulation of uPAR and cathepsin B initiates caspase-dependent mitochondrial apoptosis in U251 cells and caspase-independent mitochondrial apoptosis in 5310 cells. Thus, targeting uPAR and cathepsin B-mediated signaling using siRNA may serve as a novel therapeutic strategy for the treatment of gliomas.
Project description:Geranylated 4-phenylcoumarins DMDP-1 and DMDP-2 isolated from Mesua elegans were elucidated for their role in inducing caspase-independent programmed cell death (CI-PCD) in prostate cancer cell lines, PC-3 and DU 145, respectively. Cell homeostasis disruption was demonstrated upon treatment, as shown by the increase in calcium ion through colourimetric assay and endoplasmic reticulum (ER) stress markers GRP 78 and p-eIF2? through western blot. Subsequently, cytoplasmic death protease calpain-2 also showed increased activity during DMDP-1 & -2 treatments, while lysosomic death protease cathepsin B activity was significantly increased in PC-3 treated with DMDP-1. Flow cytometry showed a reduction in mitochondrial membrane potential in both cell lines, while western blotting showed translocation of mitochondrial death protease AIF into the cytoplasm in its truncated form. Furthermore, DMDP-1 & -2 treatments caused significant increase in superoxide level and oxidative DNA damage. Concurrent inhibition of calpain-2 and cathepsin B during the treatment showed an attenuation of cell death in both cell lines. Hence, DMDP-1 & -2 induce CI-PCD in prostate cancer cell lines through calpain-2 and cathepsin B.