Role of the unfolded protein response in cell death

Unfolded protein response (UPR) is an important genomic response to endoplasmic reticulum (ER) stress. The ER chaperones, GRP78 and Gadd153, play critical roles in cell survival or cell death as part of the UPR, which is regulated by three signaling pathways: PERK/ATF4, IRE1/XBP1 and ATF6. During the UPR, accumulated unfolded protein is either correctly refolded, or unsuccessfully refolded and degraded by the ubiquitin-proteasome pathway. When the unfolded protein exceeds a threshold, damaged cells are committed to cell death, which is mediated by ATF4 and ATF6, as well as activation of the JNK/AP-1/Gadd153-signaling pathway. Gadd153 suppresses activation of Bcl-2 and NF-κB. UPR-mediated cell survival or cell death is regulated by the balance of GRP78 and Gadd153 expression, which is coregulated by NF-κB in accordance with the magnitude of ER stress. Less susceptibility to cell death upon activation of the UPR may contribute to tumor progression and drug resistance of solid tumors.     

Deficiency in the mitochondrial apoptotic pathway reveals the toxic potential of autophagy under ER stress conditions

Endoplasmic reticulum (ER) stress-induced cell death is normally associated with activation of the mitochondrial apoptotic pathway, which is characterized by CYCS (cytochrome c, somatic) release, apoptosome formation, and caspase activation, resulting in cell death. In this study, we demonstrate that under conditions of ER stress cells devoid of CASP9/caspase-9 or BAX and BAK1, and therefore defective in the mitochondrial apoptotic pathway, still undergo a delayed form of cell death associated with the activation of caspases, therefore revealing the existence of an alternative stress-induced caspase activation pathway. We identified CASP8/caspase-8 as the apical protease in this caspase cascade, and found that knockdown of either of the key autophagic genes, ATG5 or ATG7, impacted on CASP8 activation and cell death induction, highlighting the crucial role of autophagy in the activation of this novel ER stress-induced death pathway. In line with this, we identified a protein complex composed of ATG5, FADD, and pro-CASP8 whose assembly coincides with caspase activation and cell death induction. Together, our results reveal the toxic potential of autophagy in cells undergoing ER stress that are defective in the mitochondrial apoptotic pathway, and suggest a model in which the autophagosome functions as a platform facilitating pro-CASP8 activation. Chemoresistance, a common problem in the treatment of cancer, is frequently caused by the downregulation of key mitochondrial death effector proteins. Alternate stress-induced apoptotic pathways, such as the one described here, may become of particular relevance for tackling the problem of chemoresistance in cancer cells.     

Deficiency in the mitochondrial apoptotic pathway reveals the toxic potential of autophagy under ER stress conditions

Endoplasmic reticulum (ER) stress-induced cell death is normally associated with activation of the mitochondrial apoptotic pathway, which is characterized by CYCS (cytochrome c, somatic) release, apoptosome formation, and caspase activation, resulting in cell death. In this study, we demonstrate that under conditions of ER stress cells devoid of CASP9/caspase-9 or BAX and BAK1, and therefore defective in the mitochondrial apoptotic pathway, still undergo a delayed form of cell death associated with the activation of caspases, therefore revealing the existence of an alternative stress-induced caspase activation pathway. We identified CASP8/caspase-8 as the apical protease in this caspase cascade, and found that knockdown of either of the key autophagic genes, ATG5 or ATG7, impacted on CASP8 activation and cell death induction, highlighting the crucial role of autophagy in the activation of this novel ER stress-induced death pathway. In line with this, we identified a protein complex composed of ATG5, FADD, and pro-CASP8 whose assembly coincides with caspase activation and cell death induction. Together, our results reveal the toxic potential of autophagy in cells undergoing ER stress that are defective in the mitochondrial apoptotic pathway, and suggest a model in which the autophagosome functions as a platform facilitating pro-CASP8 activation. Chemoresistance, a common problem in the treatment of cancer, is frequently caused by the downregulation of key mitochondrial death effector proteins. Alternate stress-induced apoptotic pathways, such as the one described here, may become of particular relevance for tackling the problem of chemoresistance in cancer cells.     

Plant ERD2-like proteins function as endoplasmic reticulum luminal protein receptors and participate in programmed cell death during innate immunity

Plant secondary metabolites, such as those derived from the phenylpropanoid pathway, have a beneficial effect on human health. Manipulation of metabolic flux in the phenylpropanoid pathway is important for achieving enhanced production of compounds such as anthocyanins, flavonoids and isoflavonoids. Here, we describe the development of a high-throughput molecular evolution approach that can be used for catalytic improvement of at least four key phenylpropanoid pathway enzymes, within the context of the metabolic pathway. This method uses yeast cells that express plant phenylpropanoid pathway enzymes, leading to formation of a colored intermediate that can be used as a readout in high-throughput screening. Here we report the identification of improved tomato peel 4-coumarate:CoA ligase variants using this approach. We found that the wild-type enzyme is strongly allosterically inhibited by naringenin, a downstream product of the pathway. Surprisingly, at least two of the improved variants are completely insensitive to feedback inhibition by naringenin. We suggest that this inhibition is exerted through a unique and previously unrecognized allosteric domain.     

Peroxidative stress selectively down-regulates the neuronal stress response activated under conditions of endoplasmic reticulum dysfunction

Oxidative stress has been implicated in mechanisms leading to neuronal cell injury in various pathological states of the brain. Here, we investigated the effect of peroxide exposure on the expression of genes coding for cytoplasmic and endoplasmic reticulum (ER) stress proteins. Primary neuronal cell cultures were exposed to H(2)O(2) for 6 h and mRNA levels of hsp70, grp78, grp94, gadd153 were evaluated by quantitative PCR. In addition, peroxide-induced changes in protein synthesis and cell viability were investigated. Peroxide treatment of cells triggered an almost 12-fold increase in hsp70 mRNA levels, but a significant decrease in grp78, grp94 and gadd153 mRNA levels. To establish whether peroxide exposure blocks the ER-resident stress response, cells were also exposed to thapsigargin (Tg, a specific inhibitor of ER Ca(2+)-ATPase) which has been shown to elicit the ER stress response. Tg exposure induced 7.2-fold, 3.6-fold and 8.8-fold increase in grp78, grp94 and gadd153 mRNA levels, respectively. However, after peroxide pre-exposure, the Tg-induced effect on grp78, grp94 and gadd153 mRNA levels was completely blocked. The results indicate that oxidative damage causes a selective down-regulation of the neuronal stress response activated under conditions of ER dysfunction. This down-regulation was only observed in cultures exposed to peroxide levels which induced severe suppression of protein synthesis and cell injury, implying a causative link between peroxide-induced down-regulation of ER stress response system and development of neuronal cell injury. These observations could have implications for our understanding of the mechanisms underlying neuronal cell injury in pathological states of the brain associated with oxidative damage, including Alzheimer's disease where the neuronal stress response activated under conditions of ER dysfunction has been shown to be down-regulated. Down-regulation of ER stress response may increase the sensitivity of neurones to an otherwise nonlethal form of stress.     

Coupling endoplasmic reticulum stress to the cell death program in mouse melanoma cells: effect of curcumin

The microenvironment of cancerous cells includes endoplasmic reticulum (ER) stress the resistance to which is required for the survival and growth of tumors. Acute ER stress triggers the induction of a family of ER stress proteins that promotes survival and/or growth of the cancer cells, and also confers resistance to radiation and chemotherapy. Prolonged or severe ER stress, however, may ultimately overwhelm the cellular protective mechanisms, triggering cell death through specific programmed cell death (pcd) pathways. Thus, downregulation of the protective stress proteins may offer a new therapeutic approach to cancer treatment. In this regard, recent reports have demonstrated the roles of the phytochemical curcumin in the inhibition of proteasomal activity and triggering the accumulation of cytosolic Ca²⁺ by inhibiting the Ca²⁺-ATPase pump, both of which enhance ER stress. Using a mouse melanoma cell line, we investigated the possibility that curcumin may trigger ER stress leading to programmed cell death. Our studies demonstrate that curcumin triggers ER stress and the activation of specific cell death pathways that feature caspase cleavage and activation, p23 cleavage, and downregulation of the anti-apoptotic Mcl-1 protein.     

A new role for BiP: closing the aqueous translocon pore during protein integration into the ER membrane.

In mammalian cells, most membrane proteins are inserted cotranslationally into the ER membrane at sites termed translocons. Although each translocon forms an aqueous pore, the permeability barrier of the membrane is maintained during integration, even when the otherwise tight ribosome-translocon seal is opened to allow the cytoplasmic domain of a nascent protein to enter the cytosol. To identify the mechanism by which membrane integrity is preserved, nascent chain exposure to each side of the membrane was determined at different stages of integration by collisional quenching of a fluorescent probe in the nascent chain. Comparing integration intermediates prepared with intact, empty, or BiP-loaded microsomes revealed that the lumenal end of the translocon pore is closed by BiP in an ATP-dependent process before the opening of the cytoplasmic ribosome-translocon seal during integration. This BiP function is distinct from its previously identified role in closing ribosome-free, empty translocons because of the presence of the ribosome at the translocon and the nascent membrane protein that extends through the translocon pore and into the lumen during integration. Therefore, BiP is a key component in a sophisticated mechanism that selectively closes the lumenal end of some, but not all, translocons occupied by a nascent chain. By using collisional quenchers of different sizes, the large internal diameter of the ribosome-bound aqueous translocon pore was found to contract when BiP was required to seal the pore during integration. Therefore, closure of the pore involves substantial conformational changes in the translocon that are coupled to a complex sequence of structural rearrangements on both sides of the ER membrane involving the ribosome and BiP.     

Inter‐regulation of the unfolded protein response and auxin signaling

The unfolded protein response (UPR) is a signaling network triggered by overload of protein‐folding demand in the endoplasmic reticulum (ER), a condition termed ER stress. The UPR is critical for growth and development; nonetheless, connections between the UPR and other cellular regulatory processes remain largely unknown. Here, we identify a link between the UPR and the phytohormone auxin, a master regulator of plant physiology. We show that ER stress triggers down‐regulation of auxin receptors and transporters in Arabidopsis thaliana. We also demonstrate that an Arabidopsis mutant of a conserved ER stress sensor IRE1 exhibits defects in the auxin response and levels. These data not only support that the plant IRE1 is required for auxin homeostasis, they also reveal a species‐specific feature of IRE1 in multicellular eukaryotes. Furthermore, by establishing that UPR activation is reduced in mutants of ER‐localized auxin transporters, including PIN5, we define a long‐neglected biological significance of ER‐based auxin regulation. We further examine the functional relationship of IRE1 and PIN5 by showing that an ire1 pin5 triple mutant enhances defects of UPR activation and auxin homeostasis in ire1 or pin5. Our results imply that the plant UPR has evolved a hormone‐dependent strategy for coordinating ER function with physiological processes.     

Caspase-like protease involvement in the control of plant cell death

Cell death as a highly regulated process has now been recognized to be an important, if not essential, pathway that is ubiquitous in all multicellular eukaryotes. In addition to playing key roles in the morphogenesis and sculpting of the organs to give rise to highly specialized forms and shapes, cell death also participates in the programmed creation of specialized cell types for essential functions such as the selection of B cells in the immune system of mammals and the formation of tracheids in the xylem of vascular plants. Studies of apoptosis, the most well-characterized form of animal programmed cell death, have culminated in the identification of a central tripartite death switch the enzymatic component of which is a conserved family of cysteine proteases called caspases. Studies in invertebrates and other animal models suggest that caspases are conserved regulators of apoptotic cell death in all metazoans. In plant systems, the identities of the main executioners that orchestrate cell death remain elusive. Recent evidence from inhibitor studies and biochemical approaches suggests that caspase-like proteases may also be involved in cell death control in higher plants. Furthermore, the mitochondrion and reactive oxygen species may well constitute a common pathway for cell death activation in both animal and plant cells. Cloning of plant caspase-like proteases and elucidation of the mechanisms through which mitochondria may regulate cell death in both systems should shed light on the evolution of cell death control in eukaryotes and may help to identify essential components that are highly conserved in eukaryotes.     

p25/cyclin-dependent kinase 5 promotes the progression of cell death in nucleus of endoplasmic reticulum-stressed neurons

Dysregulation of cyclin-dependent kinase 5 (Cdk5) by cleavage of its activator p35 to p25 by calpain is involved in the neuronal cell death observed in neurodegenerative disorders, including Alzheimer's disease. However, it is not yet clear how p25/Cdk5 induces cell death, although its cytosolic localization or extended half life are thought to be involved. We show here that endoplasmic reticulum (ER) stress causes the calpain-dependent cleavage of p35 to p25 in primary cultured cortical neurons. Generation of p25 occurred at a cell death execution step in ER-stressed neurons. p25 translocated to the nucleus in ER-stressed neurons, whereas p35/Cdk5 was perinuclear in control neurons. Cdk5 inhibitors or dominant-negative Cdk5 suppressed ER stress-induced neuronal cell death. These findings indicate that p25/Cdk5 is a proapoptotic factor that promotes ER stress-induced neuronal cell death in nuclei.     

The unfolded protein response in human corneal endothelial cells following hypothermic storage: implications of a novel stress pathway

Human corneal endothelial cells (HCEC) have become increasingly important for a range of eye disease treatment therapies. Accordingly, a more detailed understanding of the processing and preservation associated stresses experienced by corneal cells might contribute to improved therapeutic outcomes. To this end, the unfolded protein response (UPR) pathway was investigated as a potential mediator of corneal cell death in response to hypothermic storage. Once preservation-induced failure had begun in HCECs stored at 4 °C, it was noted that necrosis accounted for the majority of cell death but with significant apoptotic involvement, peaking at several hours post-storage (4–8 h). Western blot analysis demonstrated changes associated with apoptotic activation (caspase 9, caspase 3, and PARP cleavage). Further, the activation of the UPR pathway was observed through increased and sustained levels of ER folding and chaperone proteins (Bip, PDI, and ERO1-Lα) in samples experiencing significant cell death. Modulation of the UPR pathway using the specific inhibitor, salubrinal, resulted in a 2-fold increase in cell survival in samples experiencing profound cold-induced failure. Furthermore, this increased cell survival was associated with increased membrane integrity, cell attachment, and decreased necrotic cell death populations. Conversely, addition of the UPR inducer, tunicamycin, during cold exposure resulted in a significant decrease in HCEC survival during the recovery period. These data implicate for the first time that this novel cell stress pathway may be activated in HCEC as a result of the complex stresses associated with hypothermic exposure. The data suggest that the targeted control of the UPR pathway during both processing and preservation protocols may improve cell survival and function of HCEC thus improving the clinical utility of these cells as well as whole human corneas.     

Evaluating endoplasmic reticulum stress and unfolded protein response through the lens of ecology and evolution

Considerable progress has been made in understanding the physiological basis for variation in the life-history patterns of animals, particularly with regard to the roles of oxidative stress and hormonal regulation. However, an underappreciated and understudied area that could play a role in mediating inter- and intraspecific variation of life history is endoplasmic reticulum (ER) stress, and the resulting unfolded protein response (UPR^ER). ER stress response and the UPR^ER maintain proteostasis in cells by reducing the intracellular load of secretory proteins and enhancing protein folding capacity or initiating apoptosis in cells that cannot recover. Proper modulation of the ER stress response and execution of the UPR^ER allow animals to respond to intracellular and extracellular stressors and adapt to constantly changing environments. ER stress responses are heritable and there is considerable individual variation in UPR^ER phenotype in animals, suggesting that ER stress and UPR^ER phenotype can be subjected to natural selection. The variation in UPR^ER phenotype presumably reflects the way animals respond to ER stress and environmental challenges. Most of what we know about ER stress and the UPR^ER in animals has either come from biomedical studies using cell culture or from experiments involving conventional laboratory or agriculturally important models that exhibit limited genetic diversity. Furthermore, these studies involve the assessment of experimentally induced qualitative changes in gene expression as opposed to the quantitative variations that occur in naturally existing populations. Almost all of these studies were conducted in controlled settings that are often quite different from the conditions animals experience in nature. Herein, we review studies that investigated ER stress and the UPR^ER in relation to key life-history traits including growth and development, reproduction, bioenergetics and physical performance, and ageing and senescence. We then ask if these studies can inform us about the role of ER stress and the UPR^ER in mediating the aforementioned life-history traits in free-living animals. We propose that there is a need to conduct experiments pertaining to ER stress and the UPR^ER in ecologically relevant settings, to characterize variation in ER stress and the UPR^ER in free-living animals, and to relate the observed variation to key life-history traits. We urge others to integrate multiple physiological systems and investigate how interactions between ER stress and oxidative stress shape life-history trade-offs in free-living animals.     

Identification of the viroid-induced tomato pathogenesis-related (PR) protein P23 as the thaumatin-like tomato protein NP24 associated with osmotic stress

P23, a 23 kDa pathogenesis-related (PR) protein, was purified from citrus exocortis viroid (CEVd)-infected tomato leaves. Partial amino acid sequencing of this protein including the N-terminal and nine additional tryptic fragments covering about 50% of its primary structure revealed extensive homologies to the members of the family of plant thaumatin-like proteins. Sequence alignment revealed that tomato P23 is the previously described NP24 protein found to be associated to osmotic stress in tomato. In view of this fact the possible role of pathogenesis-related P23 protein as a component of a general mechanism of response of the plant is discussed.     

M(en)TORship lessons on life and death by the integrated stress response

Cells employ pro-survival and pro-adaptive pathways to cope with different forms of environmental stress. When stress is excessive, and the damage caused by it is unsustainable, cells engage pro-death pathways, which are in place to protect the host from the deleterious effects of harmed cells. Two important pathways that determine the balance between survival and death of stressed cells are the integrated stress response (ISR) and the mammalian target of rapamycin (mTOR), both of which converge at the level of mRNA translation. The two pathways have established avenues of communication to control their activity and determine the fate of stressed cells in a context-dependent manner. The functional interplay between the ISR and mTOR may have significant ramifications in the development and treatment of human diseases such as diabetes, neurodegeneration and cancer.     

A novel patatin-like protein from cotton plant,GhPat1, is co-expressed with GhLox1 during <Emphasis Type="Italic">Xanthomonas campestris</Emphasis>-mediated hypersensitive cell death

In cotton plant, Xanthomonas-induced hypersensitive response (HR) is accompanied by a lipid peroxidation process involving a 9-lipoxygenase (LOX), GhLox1. Initiation of this oxidative metabolism implies the release of the LOX substrates, or polyunsaturated fatty acids. Since patatin-like proteins (PLPs) are likely candidates for mediating the latter step, we searched for genes encoding such enzymes, identified and cloned one of them that we named GhPat1. Biochemical and molecular studies showed that GhPat1 expression was up-regulated during the incompatible interaction, prior to the onset of the corresponding galactolipase activity and cell death symptoms in tissues. Protein sequence analysis and modelling also revealed that GhPat1 catalytic amino acids and fold were conserved across plant PLPs. Based on these results and our previous work (Jalloul et al. in Plant J 32:1-12, 2002), a role for GhPat1, in synergy with GhLox1, during HR-specific lipid peroxidation is discussed.