Modulating stress responses by the UPRosome: a matter of life and death

The accumulation of unfolded proteins in the endoplasmic reticulum (ER) triggers the unfolded protein response (UPR) through the activation of specialized sensors including inositol-requiring enzyme-1α (IRE1α). IRE1α signals by assembling a dynamic protein platform referred to as the UPRosome, where different modulator and adaptor proteins assemble to regulate the kinetics and amplitude of UPR effector responses. Conversely, chronic ER stress can cause apoptosis. Recent evidence indicates that several apoptosis-related proteins interact with IRE1α, regulating its prosurvival activities and performing a dual function in the regulation of cell death and adaptation to stress. Based on the increasing relevance of ER stress to the occurrence of diverse pathological conditions, strategies to target and modulate the assembly and composition of the UPRosome could have therapeutic benefits for disease intervention.     

Study on the effect of IRE1α on cell growth and apoptosis via modulation PLK1 in ER stress response

The mammalian unfolded protein response (UPR) protects the cell against the stress of misfolded proteins in the endoplasmic reticulum (ER). Failure to adapt to ER stress causes the UPR to trigger apoptosis. Inositol-requiring enzyme-1a (IRE1a), as one of three unfolded protein sensors in UPR signaling pathways, senses ER unfolded proteins through an ER lumenal domain that becomes oligomerized during ER stress. It is known to be important for ER stress-mediated apoptosis and cell growth, but the exact molecular mechanism underlying these processes remains unexplored. In this study, we report that knockdown of IRE1a by an siRNA silencing approach enhanced, whereas its overexpression inhibited, cell proliferation in Hepatoma cells. Besides, overexpression of IRE1a induced, while its repression inhibited, ER stress-mediated apoptosis in Hepatomas cells. Furthermore, we found that overexpressed IRE1a can down-regulate Polo-like kinase 1(PLK1) from mRNA and protein two levels. IRE1a-mediated induction of apoptosis and inhibition of proliferation in response to ER stress is through downregulation PLK1, an early trigger for G2/M transition known to be participated in regulating cell proliferation and cell apoptosis. Collectively, these findings reveal a novel critical role of IRE1a in ER stress-mediated apoptosis and the molecular mechanisms involved. IRE1a may be a useful molecular target for the development of novel predictive and therapeutic strategies in cancer.     

IRE1α induces thioredoxin-interacting protein to activate the NLRP3 inflammasome and promote programmed cell death under irremediable ER stress

When unfolded proteins accumulate to irremediably high levels within the endoplasmic reticulum (ER), intracellular signaling pathways called the unfolded protein response (UPR) become hyperactivated to?cause programmed cell death. We discovered that?thioredoxin-interacting protein (TXNIP) is?a critical node in this "terminal UPR." TXNIP becomes rapidly induced by IRE1α, an ER bifunctional kinase/endoribonuclease (RNase). Hyperactivated IRE1α increases TXNIP mRNA stability by reducing levels of a TXNIP destabilizing microRNA, miR-17. In turn, elevated TXNIP protein activates the NLRP3 inflammasome, causing procaspase-1 cleavage and interleukin 1β (IL-1β) secretion. Txnip gene deletion reduces pancreatic β cell death during ER stress and suppresses diabetes caused by proinsulin misfolding in the Akita mouse. Finally, small molecule?IRE1α RNase inhibitors suppress TXNIP production to block IL-1β secretion. In summary, the IRE1α-TXNIP pathway is used in the terminal UPR to promote sterile inflammation and programmed cell death and may be targeted to develop effective treatments for cell degenerative diseases.     

The unknown face of IRE1α – Beyond ER stress

IRE1α (Inositol Requiring kinase Enzyme 1 alpha), a transmembrane protein localized to the endoplasmic reticulum (ER) is a master regulator of the unfolded protein response (UPR) pathway. The fate determining steps during ER stress-induced apoptosis are greatly attributed to IRE1α’s endoribonuclease and kinase activities. Apart from its role as a chief executioner in ER stress, recent studies have shown that upon activation in the presence or absence of ER stress, IRE1α executes multiple cellular processes such as differentiation, immune response, progression and repression of the cell cycle. Besides its crucial role in protein misfolding, the versatile contributions of IRE1α in other cellular functions are greatly unknown. In this review, we have discussed the structural conservation of IRE1 among eukaryotes, the mechanisms underlying its activation and the recent understandings of the non-apoptotic functions of IRE1 other than ER stress-induced cell death.     

Tumor necrosis factor alpha (TNFalpha) induces the unfolded protein response (UPR) in a reactive oxygen species (ROS)-dependent fashion, and the UPR counteracts ROS accumulation by TNFalpha

Accumulation of unfolded proteins in the endoplasmic reticulum (ER) causes ER overload, resulting in ER stress. To cope with ER stress, mammalian cells trigger a specific response known as the unfolded protein response (UPR). Although recent studies have indicated cross-talk between ER stress and oxidative stress, the mechanistic link is not fully understood. By using murine fibrosarcoma L929 cells, in which tumor necrosis factor (TNF) alpha induces accumulation of reactive oxygen species (ROS) and cell death, we show that TNFalpha induces the UPR in a ROS-dependent fashion. In contrast to TNFalpha, oxidative stresses by H2O2 or arsenite only induce eukaroytic initiation factor 2alpha phosphorylation, but not activation of PERK- or IRE1-dependent pathways, indicating the specificity of downstream signaling induced by various oxidative stresses. Conversely, the UPR induced by tunicamycin substantially suppresses TNFalpha-induced ROS accumulation and cell death by inhibiting reduction of cellular glutathione levels. Collectively, some, but not all, oxidative stresses induce the UPR, and pre-emptive UPR counteracts TNFalpha-induced ROS accumulation.     

Homocysteine induces programmed cell death in human vascular endothelial cells through activation of the unfolded protein response.

Severe hyperhomocysteinemia is associated with endothelial cell injury that may contribute to an increased incidence of thromboembolic disease. In this study, homocysteine induced programmed cell death in human umbilical vein endothelial cells as measured by TdT-mediated dUTP nick end labeling assay, DNA ladder formation, induction of caspase 3-like activity, and cleavage of procaspase 3. Homocysteine-induced cell death was specific to homocysteine, was not mediated by oxidative stress, and was mimicked by inducers of the unfolded protein response (UPR), a signal transduction pathway activated by the accumulation of unfolded proteins in the lumen of the endoplasmic reticulum. Dominant negative forms of the endoplasmic reticulum-resident protein kinases IRE1alpha and -beta, which function as signal transducers of the UPR, prevented the activation of glucose-regulated protein 78/immunoglobulin chain-binding protein and C/EBP homologous protein/growth arrest and DNA damage-inducible protein 153 in response to homocysteine. Furthermore, overexpression of the point mutants of IRE1 with defective RNase more effectively suppressed the cell death than the kinase-defective mutant. These results indicate that homocysteine induces apoptosis in human umbilical vein endothelial cells by activation of the UPR and is signaled through IRE1. The studies implicate that the UPR may cause endothelial cell injury associated with severe hyperhomocysteinemia.     

FAD-linked presenilin-1 mutants impede translation regulation under ER stress

FAD mutations in presenilin-1 (PS1) cause attenuation of the induction of the endoplasmic reticulum (ER)-resident chaperone GRP78/BiP under ER stress, due to disturbed function of IRE1, the sensor for accumulation of unfolded protein in the ER lumen. PERK, an ER-resident transmembrane protein kinase, is also a sensor for the unfolded protein response (UPR), causing phosphorylation of eukaryotic initiation factor 2alpha (eIF2alpha) to inhibit translation initiation. Here, we report that the FAD mutant PS1 disturbs the UPR by attenuating both the activation of PERK and the phosphorylation of eIF2alpha. Consistent with the results of a disturbed UPR, inhibition of protein synthesis under ER stress was impaired in cells expressing PS1 mutants. These results suggest that mutant PS1 impedes general translational attenuation regulated by PERK and eIF2alpha, resulting in an increased load of newly synthesized proteins into the ER and subsequently increasing vulnerability to ER stress.     

Lack of XBP-1 Impedes Murine Cytomegalovirus Gene Expression

The unfolded protein response (UPR) is an endoplasmic reticulum (ER)-to-nucleus signaling cascade induced in response to ER stress. The UPR aims at restoring homeostasis, but can also induce apoptosis if stress persists. Infection by human and murine cytomegaloviruses (CMVs) provokes ER stress and induces the UPR. However, both CMVs manipulate the UPR to promote its prosurvival activity and delay apoptosis. The underlying mechanisms remain largely unknown. Recently, we demonstrated that MCMV and HCMV encode a late protein to target IRE1 for degradation. However, the importance of its downstream effector, X Box binding protein 1 (XBP-1), has not been directly studied. Here we show that deletion of XBP-1 prior to or early after infection confers a transient delay in viral propagation in fibroblasts that can be overcome by increasing the viral dose. A similar phenotype was demonstrated in peritoneal macrophages. In vivo, acute infection by MCMV is reduced in the absence of XBP-1. Our data indicate that removal of XBP-1 confers a kinetic delay in early stages of MCMV infection and suggest that the late targeting of IRE1 is aimed at inhibiting activities other than the splicing of XBP-1 mRNA.     

USP14 inhibits ER-associated degradation via interaction with IRE1α

Accumulation of unfolded proteins within the endoplasmic reticulum (ER) lumen induces ER stress. Eukaryotic cells possess the ER quality control systems, the unfolded protein response (UPR), to adapt to ER stress. IRE1α is one of the ER stress receptors and mediates the UPR. Here, we identified ubiquitin specific protease (USP) 14 as a binding partner of IRE1α. USP14 interacted with the cytoplasmic region of IRE1α, and the endogenous interaction between USP14 and IRE1α was inhibited by ER stress. Overexpression of USP14 inhibited the ER-associated degradation (ERAD) pathway, and USP14 depletion by small interfering RNA effectively activated ERAD. These findings suggest that USP14 is a novel player in the UPR by serving as a physiological inhibitor of ERAD under the non-stressed condition.     

Cab45S inhibits the ER stress-induced IRE1-JNK pathway and apoptosis via GRP78/BiP

Disturbance of endoplasmic reticulum (ER) homeostasis causes ER stress and leads to activation of the unfolded protein response, which reduces the stress and promotes cell survival at the early stage of stress, or triggers cell death and apoptosis when homeostasis is not restored under prolonged ER stress. Here, we report that Cab45S, a member of the CREC family, inhibits ER stress-induced apoptosis. Depletion of Cab45S increases inositol-requiring kinase 1 (IRE1) activity, thus producing more spliced forms of X-box-binding protein 1 mRNA at the early stage of stress and leads to phosphorylation of c-Jun N-terminal kinase, which finally induces apoptosis. Furthermore, we find that Cab45S specifically interacts with 78-kDa glucose-regulated protein/immunoglobulin heavy chain binding protein (GRP78/BiP) on its nucleotide-binding domain. Cab45S enhances GRP78/BiP protein level and stabilizes the interaction of GRP78/BiP with IRE1 to inhibit ER stress-induced IRE1 activation and apoptosis. Together, Cab45S, a novel regulator of GRP78/BiP, suppresses ER stress-induced IRE1 activation and apoptosis by binding to and elevating GRP78/BiP, and has a role in the inhibition of ER stress-induced apoptosis.     

Sustained IRE1 and ATF6 signaling is important for survival of melanoma cells undergoing ER stress

Apoptosis triggered by endoplasmic reticulum (ER) stress is associated with rapid attenuation of the IRE1α and ATF6 pathways but persistent activation of the PERK branch of the unfolded protein response (UPR) in cells. However, melanoma cells are largely resistant to ER stress-induced apoptosis, suggesting that the kinetics and durations of activation of the UPR pathways are deregulated in melanoma cells undergoing ER stress. We show here that the IRE1α and ATF6 pathways are sustained along with the PERK signaling in melanoma cells subjected to pharmacological ER stress, and that this is, at least in part, due to increased activation of the MEK/ERK pathway. In contrast to an initial increase followed by rapid reduction in activation of IRE1α and ATF6 signaling in control cells that were relatively sensitive to ER stress-induced apoptosis, activation of IRE1α and ATF6 by the pharmacological ER stress inducer tunicamycin (TM) or thapsigargin (TG) persisted in melanoma cells. On the other hand, the increase in PERK signaling lasted similarly in both types of cells. Sustained activation of IRE1α and ATF6 signaling played an important role in protecting melanoma cells from ER stress-induced apoptosis, as interruption of IRE1α or ATF6 rendered melanoma cells sensitive to apoptosis induced by TM or TG. Inhibition of MEK partially blocked IRE1α and ATF6 activation, suggesting that MEK/ERK signaling contributed to sustained activation of IRE1α and ATF6. Taken together, these results identify sustained activation of the IRE1α and ATF6 pathways of the UPR driven by the MEK/ERK pathway as an important protective mechanism against ER stress-induced apoptosis in melanoma cells.