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.     

Roles of mitochondrial unfolded protein response in mammalian stem cells

The mitochondrial unfolded protein response (UPR^mt) is an evolutionarily conserved adaptive mechanism for improving cell survival under mitochondrial stress. Under physiological and pathological conditions, the UPR^mt is the key to maintaining intracellular homeostasis and proteostasis. Important roles of the UPR^mt have been demonstrated in a variety of cell types and in cell development, metabolism, and immune processes. UPR^mt dysfunction leads to a variety of pathologies, including cancer, inflammation, neurodegenerative disease, metabolic disease, and immune disease. Stem cells have a special ability to self-renew and differentiate into a variety of somatic cells and have been shown to exist in a variety of tissues. These cells are involved in development, tissue renewal, and some disease processes. Although the roles and regulatory mechanisms of the UPR^mt in somatic cells have been widely reported, the roles of the UPR^mt in stem cells are not fully understood. The roles and functions of the UPR^mt depend on stem cell type. Therefore, this paper summarizes the potential significance of the UPR^mt in embryonic stem cells, tissue stem cells, tumor stem cells, and induced pluripotent stem cells. The purpose of this review is to provide new insights into stem cell differentiation and tumor pathogenesis.     

Ubiquitin-like protein 5 positively regulates chaperone gene expression in the mitochondrial unfolded protein response

Perturbation of the protein-folding environment in the mitochondrial matrix selectively upregulates the expression of nuclear genes encoding mitochondrial chaperones. To identify components of the signal transduction pathway(s) mediating this mitochondrial unfolded protein response (UPR(mt)), we first isolated a temperature-sensitive mutation (zc32) that conditionally activates the UPR(mt) in C. elegans and subsequently searched for suppressors by systematic inactivation of genes. RNAi of ubl-5, a gene encoding a ubiquitin-like protein, suppresses activation of the UPR(mt) markers hsp-60::gfp and hsp-6::gfp by the zc32 mutation and by other manipulations that promote mitochondrial protein misfolding. ubl-5 (RNAi) inhibits the induction of endogenous mitochondrial chaperone encoding genes hsp-60 and hsp-6 and compromises the ability of animals to cope with mitochondrial stress. Mitochondrial morphology and assembly of multi-subunit mitochondrial complexes of biotinylated proteins are also perturbed in ubl-5(RNAi) worms, indicating that UBL-5 also counteracts physiological levels of mitochondrial stress. Induction of mitochondrial stress promotes accumulation of GFP-tagged UBL-5 in nuclei of transgenic worms, suggesting that UBL-5 effects a nuclear step required for mounting a response to the threat of mitochondrial protein misfolding.     

Sphingolipids and the unfolded protein response

The unfolded protein response (UPR) is a response by the endoplasmic reticulum to stress, classically caused by any disruption to cell homeostasis that results in an accumulation in unfolded proteins. However, there is an increasing body of research demonstrating that the UPR can also be activated by changes in lipid homeostasis, including changes in sphingolipid metabolism. Sphingolipids are a family of bioactive lipids with important roles in both the formation and integrity of cellular membranes, and regulation of key cellular processes, including cell proliferation and apoptosis. Bi-directional interactions between sphingolipids and the UPR have now been observed in a range of diseases, including cancer, diabetes and liver disease. Determining how these two key cellular components influence each other could play an important role in deciphering the causes of these diseases and potentially reveal new therapeutic approaches.     

The unfolded protein response

The unfolded protein response (UPR) is a signal transduction network activated by inhibition of protein folding in the endoplasmic reticulum (ER). The UPR coordinates adaptive responses to this stress situation, including induction of ER resident molecular chaperone and protein foldase expression to increase the protein folding capacity of the ER, induction of phospholipid synthesis, attenuation of general translation, and upregulation of ER-associated degradation to decrease the unfolded protein load of the ER, and an antioxidant response. Upon severe or prolonged ER stress the UPR induces apoptosis to eliminate unhealthy cells from an organism or a population. In this review, I will summarize our current knowledge about signal transduction pathways involved in transducing the unfolded protein signal from the ER to the nucleus or the cytosol.     

Yokukansan inhibits neuronal death during ER stress by regulating the unfolded protein response

Background

Recently, several studies have reported Yokukansan (Tsumura TJ-54), a traditional Japanese medicine, as a potential new drug for the treatment of Alzheimer''s disease (AD). Endoplasmic reticulum (ER) stress is known to play an important role in the pathogenesis of AD, particularly in neuronal death. Therefore, we examined the effect of Yokukansan on ER stress-induced neurotoxicity and on familial AD-linked presenilin-1 mutation-associated cell death.

Methods

We employed the WST-1 assay and monitored morphological changes to evaluate cell viability following Yokukansan treatment or treatment with its components. Western blotting and PCR were used to observe the expression levels of GRP78/BiP, caspase-4 and C/EBP homologous protein.

Results

Yokukansan inhibited neuronal death during ER stress, with Cnidii Rhizoma (Senkyu), a component of Yokukansan, being particularly effective. We also showed that Yokukansan and Senkyu affect the unfolded protein response following ER stress and that these drugs inhibit the activation of caspase-4, resulting in the inhibition of ER stress-induced neuronal death. Furthermore, we found that the protective effect of Yokukansan and Senkyu against ER stress could be attributed to the ferulic acid content of these two drugs.

Conclusions

Our results indicate that Yokukansan, Senkyu and ferulic acid are protective against ER stress-induced neuronal cell death and may provide a possible new treatment for AD.     

Compromising the unfolded protein response induces autophagy-mediated cell death in multiple myeloma cells

Objective

To determine whether the Unfolded Protein Response (UPR) sensors (PERK, ATF6 and IRE-1) can be targeted to promote death of Multiple Myeloma (MM) cells.

Methods

We have knocked-down separately each UPR stress sensor in human MM cell lines using RNA interference and followed MM cell death by monitoring the membrane, mitochondrial and nuclear alterations. Involvement of caspases in MM cell death consecutive to UPR sensor knock-down was analyzed by western blotting, measurement of their enzymatic activity using fluorigenic substrates and susceptibility to a pan-caspase inhibitor. Activation of the autophagic process was measured directly by detection of autophagosomes (electronic microscopy), monodansylcadaverine staining, production of the cleaved form of the microtubule-associated protein 1A/1B light chain 3 (LC3) and indirectly by analyzing the impact of pharmacological inhibitors of autophagy such as 3MA and bafilomycin A1.

Results

We show that extinction of a single UPR stress sensor (PERK) induces a non-apoptotic form of cell death in MM cells that requires autophagy for its execution. We also show that this cytotoxic autophagic process represses the apoptosis program by reducing the cytosolic release of the apoptogenic factors Smac/DIABLO and cytochrome c.

Interpretation

Altogether our findings suggest that autophagy can contribute to execution of death in mammalian cells that are exposed to mild ER stress. They also suggest that the autophagic process can regulate the intrinsic apoptotic pathway by inhibiting production of death effectors by the mitochondria, thus preventing formation of a functional apoptosome. Altogether these findings give credit to the idea that UPR sensors can be envisaged as therapeutic targets for the treatment of MM.     

Putting the brakes on the unfolded protein response

The unfolded protein response is an ancient cellular pathway for rapidly responding to endoplasmic reticulum stress. Two studies in this issue (Rubio et al. 2011. J. Cell. Biol. doi:10.1083/jcb.201007077 and Chawla et al. 2011. J. Cell. Biol. doi:10.1083/jcb.201008071) provide insight into how the unfolded protein response is tamped down to restore normal endoplasmic reticulum function. Although both papers implicate the Ire1 kinase domain as the key effector of the off-switch mechanism, alternate models for how this is achieved are proposed.     

ER stress and the unfolded protein response

Conformational diseases are caused by mutations altering the folding pathway or final conformation of a protein. Many conformational diseases are caused by mutations in secretory proteins and reach from metabolic diseases, e.g. diabetes, to developmental and neurological diseases, e.g. Alzheimer's disease. Expression of mutant proteins disrupts protein folding in the endoplasmic reticulum (ER), causes ER stress, and activates a signaling network called the unfolded protein response (UPR). The UPR increases the biosynthetic capacity of the secretory pathway through upregulation of ER chaperone and foldase expression. In addition, the UPR decreases the biosynthetic burden of the secretory pathway by downregulating expression of genes encoding secreted proteins. Here we review our current understanding of how an unfolded protein signal is generated, sensed, transmitted across the ER membrane, and how downstream events in this stress response are regulated. We propose a model in which the activity of UPR signaling pathways reflects the biosynthetic activity of the ER. We summarize data that shows that this information is integrated into control of cellular events, which were previously not considered to be under control of ER signaling pathways, e.g. execution of differentiation and starvation programs.     

Membrane biogenesis and the unfolded protein response

In addition to serving as the entry point for newly translated polypeptides making their way through the secretory pathway, the endoplasmic reticulum (ER) also synthesizes many lipid components of the entire endomembrane system. A report published in this issue implicates a signaling pathway known to respond to ER unfolded protein load in the control of phospholipid biosynthesis by the organelle (Sriburi et al., 2004). The reasonable notion that demand for ER membrane is integrated with protein processing capacity was initially suggested by genetic analysis of yeast. The new data lend direct support for this idea and imply interesting mechanistic possibilities for how this coupling develops.     

A brief history of the discovery of the mitochondrial unfolded protein response in mammalian cells

Cells have responded to stresses which cause proteins to come out of solution by evolving unfolded protein response mechanisms. In eukaryotic cells there are several such mechanisms covering the whole cell and sub-celllular organelles. This review describes discoveries that describe the unfolded protein response in the matrix compartment of mitochondria (mtUPRR) of mammalian cells.     

Differential effects of unfolded protein response pathways on axon injury-induced death of retinal ganglion cells

Loss of retinal ganglion cells (RGCs) accounts for visual function deficits after optic nerve injury, but how axonal insults lead to neuronal death remains elusive. By using an optic nerve crush model that results in the death of the majority of RGCs, we demonstrate that axotomy induces differential activation of distinct pathways of the unfolded protein response in axotomized RGCs. Optic nerve injury provokes a sustained CCAAT/enhancer binding homologous protein (CHOP) upregulation, and deletion of CHOP promotes RGC survival. In contrast, IRE/XBP-1 is only transiently activated, and forced XBP-1 activation dramatically protects RGCs from axon injury-induced death. Importantly, such differential activations of CHOP and XBP-1 and their distinct effects on neuronal cell death are also observed in RGCs with other types of axonal insults, such as vincristine treatment and intraocular pressure elevation, suggesting a new protective strategy for neurodegeneration associated with axonal damage.     

Mitochondrial S-adenosylmethionine deficiency induces mitochondrial unfolded protein response and extends lifespan in Caenorhabditis elegans

S-adenosylmethionine (SAM), generated from methionine and ATP by S-adenosyl methionine synthetase (SAMS), is the universal methyl group donor required for numerous cellular methylation reactions. In Caenorhabditis elegans, silencing sams-1, the major isoform of SAMS, genetically or via dietary restriction induces a robust mitochondrial unfolded protein response (UPR^mt) and lifespan extension. In this study, we found that depleting SAMS-1 markedly decreases mitochondrial SAM levels. Moreover, RNAi knockdown of SLC-25A26, a carrier protein responsible for transporting SAM from the cytoplasm into the mitochondria, significantly lowers the mitochondrial SAM levels and activates UPR^mt, suggesting that the UPR^mt induced by sams-1 mutations might result from disrupted mitochondrial SAM homeostasis. Through a genetic screen, we then identified a putative mitochondrial tRNA methyltransferase TRMT-10C.2 as a major downstream effector of SAMS-1 to regulate UPR^mt and longevity. As disruption of mitochondrial tRNA methylation likely leads to impaired mitochondrial tRNA maturation and consequently reduced mitochondrial translation, our findings suggest that depleting mitochondrial SAM level might trigger UPR^mt via attenuating protein translation in the mitochondria. Together, this study has revealed a potential mechanism by which SAMS-1 regulates UPR^mt and longevity.